EP1163252A2 - Compositions, kits et procedes concernant le gene fez1 de l'homme, nouveau gene suppresseur des tumeurs - Google Patents

Compositions, kits et procedes concernant le gene fez1 de l'homme, nouveau gene suppresseur des tumeurs

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Publication number
EP1163252A2
EP1163252A2 EP00912007A EP00912007A EP1163252A2 EP 1163252 A2 EP1163252 A2 EP 1163252A2 EP 00912007 A EP00912007 A EP 00912007A EP 00912007 A EP00912007 A EP 00912007A EP 1163252 A2 EP1163252 A2 EP 1163252A2
Authority
EP
European Patent Office
Prior art keywords
fezl
cell
eez7
protein
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00912007A
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German (de)
English (en)
Other versions
EP1163252A4 (fr
Inventor
Carlo M. Croce
Hideshi Ishii
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Thomas Jefferson University
Original Assignee
Thomas Jefferson University
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Filing date
Publication date
Application filed by Thomas Jefferson University filed Critical Thomas Jefferson University
Publication of EP1163252A2 publication Critical patent/EP1163252A2/fr
Publication of EP1163252A4 publication Critical patent/EP1163252A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates generally to cancer and tumor suppressor genes. Proliferation of normal cells is thought to be regulated by growth- promoting proto-oncogenes and by growth-constraining tumor suppressor genes (Weinberg, 1991, Science 254: 1138). Genetic alterations that inactivate tumor suppressor genes or that activate proto-oncogenes free cells from growth constraints imposed by the non-altered genes, thereby enabling tumor growth. Accumulation of genetic aberrations in a cell in vivo causes the cell to proceed from a normal growth or quiescent stage, potentially through a discernable pre-neoplastic stage, to a cancerous stage in which the cell replicates abnormally quickly, and potentially spreads to body locations at which the cell is not normally found (Knudson, 1993, Proc. Natl. Acad. Sci. USA 90: 10914; Nowell, 1993, Adv. Cancer Res. 62:1).
  • allelic loss(es) on chromosome 8p. particularly at band 21 -22 are associated with various tumors, including prostate tumors, breast tumors, head and neck squamous cell carcinomas, urinary bladder carcinomas, hepatocellular carcinomas, and hematological malignancies (Kagan et al., 1995. Oncogene 1 1 :2121; Macoska et al., 1995, Cancer Res. 55:5390: Jenkins et al.. 1998, Genes Chromosom.
  • N33 and PRZrS Two candidate tumor suppressor genes, designated N33 and PRZrS (Bookstein et al., 1997, Br. J. Urol. 79(Suppl. 1):28; Bova et al.. 1996. Genomics 35:46; MacGrogan et al., 1996, Genomics 35:55; Cher et al., 1994, Genes Chromosom. Cancer 1 1 :153; Bookstein, et al.. 1994, Genomics 24:317; Fujiwara et al.,
  • N33 is located at position 8p22. near the MSR gene locus, but no point mutations in N33 have been associated with tumors.
  • PRLTS which is located at position 8p21.3-22. The frequency of alterations in this gene was, however, very low. Thus, it is unlikely that either the N33 gene or the PRLTS gene are tumor suppressor genes associated with common cancers.
  • the tumor suppressor gene(s) located at chromosome location 8p has not been identified. The failure of others to identify this gene has delayed development of diagnostic, therapeutic, and other useful methods and compositions which involve this tumor suppressor gene. The present invention enables these methods and compositions.
  • the invention relates to an isolated polynucleotide comprising a portion which anneals with high stringency with (i.e. is substantially complementary to) 20 or more, consecutive nucleotide residues of a strand of a human FEZ1 gene.
  • An exemplary human EEZ7 gene has the nucleotide sequence S ⁇ Q ID NO: 1.
  • the portion which anneals can be substantially homologous with the residues of the human EEZ7 gene or. preferably, it can be completely homologous with those residues.
  • the portion is at least substantially homologous with at least twenty residues of an exon region of the human FEZl gene, i.e. nucleotide residues 112-456, nucleotide residues 1707-2510, and nucleotide residues 4912-5550 of a strand of S ⁇ Q ID NO: 1.
  • the isolated polynucleotide of the invention comprises a portion having the nucleotide sequence of a strand of S ⁇ Q ID NO: 3, and optionally further comprises a promoter.
  • the promoter may. for example, be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
  • the isolated polynucleotide is incorporated in a nucleic acid vector or is encoded by nucleic acid which is incorporated in a nucleic acid vector.
  • the isolated polynucleotide may, for example, have a sequence homologous with a strand of S ⁇ Q ID NO: 1 , and it can be detectably labeled.
  • detectably labeled isolated polynucleotides include immobilized polynucleotides, polynucleotides linked to a protein of a protein- ligand pair, polynucleotides linked to a ligand of a protein-ligand pair, biotinylated polynucleotides.
  • polynucleotides linked to a fluorophore polynucleotides linked to a chromophore, polynucleotides linked to an enzyme, and radio-labeled polynucleotides.
  • an immobilized polynucleotide it can be immobilized on the surface of a gene chip.
  • the isolated polynucleotide of the invention is substantially purified.
  • the isolated polynucleotide of the invention need not comprise only naturally occurring bases and linkages. It may, for example, have at least two nucleotide residues linked by a non-naturally occurring linkage other than a phosphodiester linkage such as, for example, a linkage selected -from the group consisting of phosphonate, phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate. formacetal, thioformacetal, diisopropylsilyl, acetamidate. carbamate. dimethylene-sulfide (-CH 2 -S-CH 2 -).
  • dimethylene-sulfoxide (-CH 2 -SO-CH 2 -), dimethylene-sulfone (-CH,-SO 2 -CH 2 -), 2'-O-alkyl, and 2'-deoxy-2'- fluoro phosphorothioate, phosphotriester, siloxane, carbonate, carboxymethyl ester, acetamidate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate.
  • an end of the isolated polynucleotide can be nucleolytically blocked.
  • the invention also includes an isolated polynucleotide comprising a portion which has a sequence which anneals with high stringency with at least twenty consecutive nucleotide residues of a strand of SEQ ID NO: 3.
  • the invention includes a kit for amplifying a portion of a human FEZl gene.
  • the kit comprises a first isolated polynucleotide and a second isolated polynucleotide.
  • the first isolated polynucleotide comprises a portion which anneals with high stringency with at least twenty consecutive nucleotide residues of the coding strand of SEQ ID NO: 1
  • the second isolated polynucleotide comprises a portion which anneals with high stringency with at least twenty consecutive nucleotide residues of the non-coding strand of SEQ ID NO: 1.
  • the invention further includes a kit for amplifying a portion of a cDNA generated from a transcript of a human FEZ/ gene.
  • the kit comprises a first isolated polynucleotide and a second isolated polynucleotide.
  • a portion of the first isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of the coding strand of S ⁇ Q ID NO: 1
  • a portion of the second isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of the non-coding strand of S ⁇ Q ID NO: 1.
  • the invention includes an animal cell comprising an exogenous DNA molecule having a portion substantially homologous with at least nucleotide residues 112-456, nucleotide residues 1707-2510, and nucleotide residues 4912-5550 of a strand of S ⁇ Q ID NO: 1.
  • the exogenous DNA molecule further comprises a promoter operably linked with the portion, and the exogenous DNA molecule is expressed in the animal cell.
  • the invention also includes a genetically altered animal comprising a cell into which an exogenous DNA molecule has been artificially introduced.
  • the exogenous DNA molecule has a portion substantially homologous with at least the coding region of a strand of a human FEZl gene.
  • the exogenous DNA molecule may, for example, have a portion substantially homologous with at least nucleotide residues 112-456, nucleotide residues 1707-2510, and nucleotide residues 4912-5550 of a strand of SEQ ID NO: 1, or it can comprise a portion having a sequence substantially homologous with a strand of SEQ ID NO: 2.
  • the invention also relates to an isolated human Fezl protein, such as a protein having an amino acid sequence substantially, or preferably completely, homologous with SEQ ID NO: 4.
  • the protein is substantially purified.
  • the invention further includes an isolated antibody which binds specifically with human Fezl protein and a hybridoma cell which produces such antibodies.
  • the invention still further relates to a method of determining the cancerous status of a sample tissue.
  • This method comprises comparing FEZl expression in the sample tissue with EEZ7 expression in a control tissue of the same type. Decreased EEZ7 expression in the sample tissue, relative to EEZ7 expression in the control tissue, is an indication that the sample tissue is cancerous.
  • the sample tissue is a phenotypically abnormal portion of a body tissue of a human
  • the control tissue is a phenotypically normal portion of the body tissue. such as an epithelial tissue.
  • the body tissue can also, for example, be selected from the group consisting of a gastrointestinal tissue, esophagus tissue, gastric tissue, colon tissue, prostate tissue, breast tissue, a hematopoietic tissue, lung tissue, melanoma tissue, cervical tissue, and ovarian tissue.
  • FEZ7 expression in the sample tissue is compared with EEZ7 expression in the control tissue by comparing the relative amounts of an indicator in the sample tissue and in the control tissue.
  • the indicator may. for example, be selected from the group consisting of a FEZl mRNA, a cDNA prepared using a EEZ7 mRNA, a DNA prepared by amplification of either of these, and Fezl protein.
  • the invention also includes a method of determining the cancerous status of a sample tissue.
  • This method comprises comparing the nucleotide sequence of a EEZ -associated polynucleotide obtained from the sample tissue with the nucleotide sequence of a control EEZ7 -associated polynucleotide. A difference between the nucleotide sequence of the EEZ7 -associated polynucleotide obtained from the sample tissue and the nucleotide sequence of the control EEZ7 -associated polynucleotide is an indication that the sample tissue is cancerous.
  • the invention includes another method of determining the cancerous status of a human sample tissue. This method comprises comparing the length of an EEZ7 -transcript-associated polynucleotide obtained from the sample tissue with the length of a control EEZ7 -transcript-associated polynucleotide. If the length of the
  • EEZ7 -transcript-associated polynucleotide obtained from the sample tissue is less than the length of the control EEZ -transcript-associated polynucleotide, then this is an indication that the sample tissue is cancerous.
  • the invention includes yet another method of determining the cancerous status of a sample tissue.
  • This method comprises assessing EEZ7 expression in the sample tissue.
  • a substantial absence of FEZl expression in the sample tissue is an indication that the sample tissue is cancerous.
  • EEZ7 expression can be assessed, for example, by assessing the presence or substantial absence of an indicator selected from the group consisting of a EEZ7 mRNA, a cDNA prepared using a EEZ7 mRNA, a DNA prepared by amplification of either of these, and Fezl protein.
  • the invention includes yet another method of determining the cancerous status of a sample tissue.
  • This method comprises detecting abnormal splicing of a EEZ7 transcript in the sample tissue.
  • Abnormal splicing of the EEZ7 transcript is an indication that the sample tissue is cancerous.
  • Abnormal splicing of the EEZ7 transcript can be detected, for example, by assessing the ability of an exon boundary polynucleotide probe to anneal with a EEZ7 -transcript-associated polynucleotide with high stringency.
  • the exon boundary polynucleotide probe is capable of annealing with high stringency with terminal portions of two sequential EEZ7 exons when the terminal portions are adjacent, but not when the terminal portions are not adjacent.
  • the invention in another aspect, relates to a method of modulating abnormal proliferation of a human cell having an altered EEZ7 gene.
  • This method comprises providing an exogenous source of Fezl protein to the cell. Abnormal proliferation of the cell is thereby inhibited, delayed, or prevented.
  • the exogenous source of Fezl protein may, for example, be a composition comprising an isolated human Fezl protein, such as a human Fezl protein having the amino acid sequence S ⁇ Q ID NO: 4.
  • the exogenous source of Fezl protein can also be an expression vector (e.g.
  • an adenovirus vector such as one comprising a vector nucleic acid having the nucleotide sequence S ⁇ Q ID NO: 60) comprising a polynucleotide having a coding region which encodes a functional Fezl protein, such as a human EEZ7 gene having the nucleotide sequence of a strand of S ⁇ Q ID NO: 3.
  • the polynucleotide can further comprise a constitutive, inducible, or tissue-specific promoter operably linked with the coding region. When the promoter is an inducible promoter, the method further comprises administering an inducer of the inducible promoter to the cell.
  • the polynucleotide may, of course, comprise a wild-type EEZ7 promoter region.
  • the invention in still another aspect, relates to a method of preventing tumorigenesis in a human cell.
  • This method comprises providing to the cell an expression vector comprising a polynucleotide having a coding region which encodes a functional Fezl protein.
  • tumorigenesis is prevented in the cell.
  • the invention also includes a method of reversibly inducing proliferation of a cell.
  • This method comprises providing an inhibitor of FEZl expression to the interior of the cell. Proliferation of the cell is thereby induced when the inhibitor is present in the interior of the cell, but is not induced when the inhibitor is not present in the interior of the cell.
  • the inhibitor may. for example, be an isolated polynucleotide comprising a portion which anneals with high stringency with at least twenty consecutive nucleotide residues of a strand of a human EEZ7 gene.
  • the isolated polynucleotide can be delivered to the interior of the cell by administering a gene vector comprising a promoter operably linked with the isolated polynucleotide to the cell.
  • the cell can be located in the body of an animal such as a human.
  • the invention in another aspect, relates to a method of determining whether a test compound is an inducer of cell proliferation.
  • This method comprises incubating a cell which comprises a functional EEZ7 gene in the presence of the test compound and assessing expression of FEZl in the cell. If expression of FEZl in the cell is decreased, relative to expression of FEZl in a cell of the same type incubated in the absence of the test compound, then the test compound is an inducer of cell proliferation.
  • the invention also includes a method of determining whether a test compound is effective to retard abnormal proliferation of a cell having an altered EEZ7 gene. This method comprises incubating the cell in the presence of the test compound and assessing expression of FEZl in the cell. If expression of FEZl in the cell is increased, relative to expression of EEZ7 in a ceil of the same type incubated in the absence of the test compound, then the test compound is effective to retard abnormal proliferation of a cell.
  • the invention further relates to a method of determining whether Fezl protein binds with polynucleotides having a test nucleotide sequence.
  • This method comprises: a) contacting Fezl protein and a test polynucleotide having the test nucleotide sequence, and b) thereafter assessing whether a detectably labeled Fezl -polynucleotide complex is formed. At least one of the Fezl protein and the test polynucleotide is detectably labeled. Formation of the complex is an indication that Fezl protein binds with polynucleotides having the test nucleotide sequence.
  • the invention still further relates to a method of identifying an inducer of cell proliferation.
  • This method comprises: a) contacting Fezl protein and a polynucleotide with which Fezl protein binds in the presence and absence of a test compound, and b) assessing formation of a Fezl -polynucleotide complex. Decreased formation of the complex in the presence of the test compound, relative to formation of Fezl - polynucleotide complex in the absence of the test compound is an indication that the test compound is an inducer of cell proliferation.
  • the invention includes a kit for selecting an anti-cancer therapeutic compound for administration to a human afflicted with a cancer.
  • the kit comprises a plurality of candidate anti-cancer therapeutic compounds and a reagent for assessing expression of FEZl in a cell.
  • the invention also includes a method of inducing a cell to proliferate.
  • This method comprises inhibiting expression of FEZl in the cell.
  • the cell is thereby induced to proliferate.
  • the cell is a cell removed from a human.
  • the cell can thereafter be returned to the human after inhibiting expression of FEZl in the cell.
  • the cell can be a cell present in the body of a human.
  • expression of FEZl in the cell can be inhibited by providing to the interior of the cell an isolated polynucleotide comprising a portion which anneals with high stringency with at least twenty consecutive nucleotide residues of a strand of a human
  • the invention further includes an enhanced human cell culture technique.
  • This technique comprises incubating human cells according to a known human cell culture technique and inhibiting EEZ7 expression in the cells.
  • the invention still further includes a method of detecting EEZ7 expression in a sample tissue. This method comprises: a) labeling an isolated antibody which binds specifically with human Fezl protein and contacting a preparation of the isolated antibody with the sample tissue, b) thereafter rinsing the tissue sample, whereby non-specifically bound antibodies are rinsed from the tissue sample, and c) assessing the presence of labeled antibodies in the tissue sample.
  • the presence of labeled antibodies in the tissue sample is an indication that EEZ7 is expressed in the tissue sample.
  • the invention includes a method of determining whether a test compound is useful for alleviating a disorder associated with aberrant tubulin polymerization. The method comprises comparing
  • tubulin polymerization in a first assay mixture which comprises tubulin, Fezl , and the test compound
  • tubulin polymerization in a second assay mixture which comprises tubulin and Fezl, but which does not comprise the test compound.
  • a difference between (e.g. the rate or extent of) tubulin polymerization in the first and second assay mixtures is an indication that the test compound is useful for alleviating the disorder.
  • the first and second assay mixtures are substantially identical, but for the presence or absence of the test compound.
  • the disorder can.
  • a tubulin hyperpolymerization disorder or a tubulin hypopolymerization disorder such as one of a disorder associated with aberrant initiation of mitosis, a disorder associated with aberrant modulation of the rate and stage of mitosis, a disorder associated with aberrant modulation of the initiation and rate of cell proliferation, a disorder associated with aberrant modulation of the initiation and rate of cell growth, a disorder associated with aberrant modulation of cell shape, a disorder associated with aberrant modulation of cell rigidity, a disorder associated with aberrant modulation of cell motility, a disorder associated with aberrant modulation of the rate of cellular DNA replication, a disorder associated with aberrant modulation of the stage of cellular DNA replication, a disorder associated with aberrant modulation of the intracellular distribution of organelles, a disorder associated with aberrant modulating the metastatic potential of a cell, and a disorder associated with aberrant modulation of cellular transformation from a non-cancerous to a cancerous phenotype.
  • the disorder can be one of tumorigenesis, tumor survival, tumor growth, and tumor metastasis.
  • test compounds include a fragment of Fezl, a peptidomimetic of a fragment of Fezl, a fragment of tubulin, a peptidomimetic of a fragment of tubulin, a fragment of EFl- ⁇ , and a peptidomimetic of a fragment of EFl- ⁇ .
  • the invention also includes a method of determining whether a test compound is useful for alleviating a disorder associated with aberrant phosphorylation of Fezl. This method comprises comparing
  • a difference between phosphorylation of Fezl in the first and second assay mixtures is an indication that the test compound is useful for alleviating the disorder.
  • the disorder can be one selected from the group consisting of tumorigenesis. tumor survival, tumor growth, and tumor metastasis.
  • the invention includes a method of determining whether a test compound is useful for alleviating a disorder associated with aberrant phosphorylation of Fezl. This method comprises comparing
  • a difference between phosphorylation of Fezl in the first and second assay mixtures is an indication that the test compound is useful for alleviating the disorder.
  • the invention includes a method of determining whether a test compound is useful for alleviating a disorder associated with aberrant binding of Fezl with a protein with which Fezl normally binds. This method comprises comparing
  • a difference between (e.g. the rate or extent of) binding of Fezl and the protein in the first and second assay mixtures is an indication that the test compound is useful for alleviating the disorder.
  • the protein of this method include tubulin and EFl- ⁇ .
  • the disorder can, for example, be any of those recited above.
  • the invention further includes a method of determining whether a test compound is an inhibitor of cell proliferation.
  • This method comprises incubating a cell which comprises a functional EEZ7 gene in the presence of the test compound and assessing expression of FEZl in the cell. If expression of FEZl in the cell is increased, relative to expression of FEZl in a cell of the same type incubated in the absence of the test compound, then the test compound is an inhibitor of cell proliferation.
  • the invention still further includes a method of inhibiting tumorigenesis in a human, the method comprising administering to the human a compound selected from the group consisting of an inducer of FEZl gene expression, an enhancer of FEZl gene expression, a inhibitor of Fezl phosphorylation, an enhancer of phosphorylated- Fezl dephosphorylation, an agent that inhibits binding of Fezl with ⁇ Fl- ⁇ . and an agent that inhibits binding of Fezl with tubulin.
  • Figure 1 comprises Figures 1A, IB, and 1C, and each of these figures relates to loss of heterozygosity (LOH) at human chromosome 8p in primary esophageal cancer tissue samples.
  • LHO heterozygosity
  • Figure 1A is a series of representative LOH analysis results obtained using tissue samples obtained from two patients. designated E26 and E46.
  • Figures 1 Ai. 1 Aiii, 1 Av. and 1 Avii depict results from tissue obtained from patient E26.
  • Figures 1 Aii, 1 Aiv, lAvi, and lAviii depict results from tissue obtained from patient E46.
  • fluorescent PCR products were generated by amplification of DNA obtained from normal (N) and tumor (T) tissue samples from the corresponding patient, and products were separated by size.
  • the horizontal axis represents DNA fragment size
  • the vertical axis i.e. peak height
  • Figures lAi and lAii correspond to D8S264; Figures 1 Aiii and lAiv correspond to LPL; Figures lAv and lAvi correspond to D8S136; and Figures lAvii and lAviii correspond FGFR1. Several fragment sizes (in base pairs) are indicated.
  • Figure 1 B is a diagram which depicts a summary of LOH analyses described herein. Results for each patient who exhibited LOH at least at one locus are shown. Filled circles represent loss of an allele. Circles containing a cross represent non-informative results owing to homozygosity at the corresponding locus. Open circles represent retention of both alleles. Cross-hatched areas of the diagram represent regions of allele loss. Hatched areas represent regions of non-informative results within the allele-loss area. The numbers atop each column refer to individual patients. The designations beside each row refer to polymo ⁇ hic markers. The region near the marker D8S261 locus, described herein, is boxed.
  • Figure 1 C is a diagram which depicts the approximate locations of genomic contigs at 8p22 which were constructed as described herein.
  • the uppermost line depicts the location of polymorphic loci on 8p.
  • the corresponding locations of YAC contigs (open boxes) and B AC contigs (horizontal lines) are indicated below the 8p map.
  • cDNA selection and shotgun sequencing were performed on YACs and BACs identified by asterisks. Eighty-seven potentially expressed sequences were isolated and located within the contigs; the approximate locations of these sequences are indicated by designations below two-headed arrows. Underlined characters indicate sequences which are expressed in normal tissues. After expression analysis in tumor and normal tissues. 9 cDNAs (circled designations) were subjected to further analysis.
  • FIG. 2 comprises Figures 2A, 2B, 2C. and 2D.
  • the predicted Fezl amino acid sequence (SEQ ID NO: 4) is depicted in Figure 2A.
  • Figure 2A lists the predicted amino acid sequence of FEZl protein, as derived from the EEZ7 cDNA. Underlined amino acid residues represent a region homologous to the DNA-binding domain of ATF-5 protein. Double-underlined amino acid residues represent a leucine zipper motif, in which repeated leucine residues are indicated.
  • Heavily-underlined amino acid residues are residues which can be phosphorylated by either a cAMP/cGMP-dependent kinase (serine residue 29) or a tyrosine kinase-dependent kinase (tyrosine residue 67). Dashed-underlined regions represent regions having related amino acid sequence motifs. Serine and threonine residues in bold or thin dotted lines represent potential casein kinase II and protein kinase C, respectively, phosphorylation sites. -Triangles indicate exon boundaries. Asterisks represent missense or nonsense mutation sites.
  • Figure 2C is an image of SDS-PAG ⁇ results as described elsewhere herein.
  • Figure 2D is an image of Northern blot analysis results which indicate EEZ7 gene expression in normal tissues.
  • a EEZ7 ORF probe S ⁇ Q ID NO: 3
  • a beta-actin probe was used, as a control, to detect expression of the beta-actin gene.
  • the arrowhead on the left of the top panel indicates the approximate position of the 6.8 kilobase EEZ7 transcript.
  • Poly(A) + RNAs (5 micrograms) were obtained from normal (i.e.
  • lane 13 non-cancerous tissues, and loaded as follows: lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas; lane 9, spleen; lane 10, thymus; lane 1 1, prostate; lane 12. testes: lane 13,
  • R ⁇ CTIR ⁇ D SHEET (RULE 91) ovary; lane 14, small intestine; lane 15, colon; and lane 16, peripheral blood lymphocyte.
  • Figure 3 comprises Figures 3A, 3B, and 3C, and relates to alterations of the EEZ7 gene in tumor cells.
  • Figure 3 A is an image which depicts results of Northern blot analysis of FEZl gene expression in cancer cells.
  • a EEZ7 cDNA probe (upper panel) and a beta-actin probe (lower panel) were used to detect expression of the corresponding genes.
  • the arrowhead on the left side of the upper panel indicates the approximate position of the 6.8-kilobase transcript of FEZl.
  • RNAs (5 micrograms) were obtained from tumor cell lines, and loaded as follows: esophageal cancer cell lines KYS ⁇ 170 (lane 1), TE12 (lane 2), TE8 (lane 3) and TE3 (lane 4); prostate cancer cell lines DU145 (lane 5), LNCaP (lane 6), PC3 (lane 7); normal prostate (lane 8); breast cancer cell lines MB231 (lane 9), SKBr3 (lane 10), BT549 (lane 1 1), HBL100 (lane 12), MB436S (lane 13), BT20 (lane 14), MB543 (lane 15), MB175 (lane 16), MCF7 (lane 17) and T47B (lane 18); normal breast (lane 19); total RNA of normal breast (lane 20); cervical cancer cell line HeLa S3 (lane 22); chronic myelogenous leukemia cell line K562 (lane 23); lymphoblastic leukemia cell line MOLT4 (lane 24); Burkitt's lymphoma cell line Raji
  • Figure 3B comprising Figures 3Bi-3Bvi, is a series of sequence chromatograms of FEZl genes obtained from three individuals having mutated EEZ7 genes. As indicated in Figure 3Bii. a point mutation in EEZ7 (TCC/Ser — > CCC/Pro) at codon 29 was identified in a primary esophageal cancer tissue sample obtained from patient ⁇ 44. Nucleotide sequences from normal DNA from patient E44 (N) and from a BAC contig (B) are shown for comparison. A bold line overlies the altered codon.
  • R ⁇ CTIR ⁇ D SHEET RULE codon 501 was identified in prostate cancer cell line PC3, as indicated in Figure 3Bvi, in which the sequence chromatogram 3'- to 5'- direction. Repeated sequencing indicated the presence of a weak signal corresponding to guanine (G) within a large adenine (A) signal in the first nucleotide at codon 501, suggesting that a fraction of the cancer cells retained the normal EEZ7 allele.
  • Figure 3 C is an image which depicts Southern blot analysis results using the EEZ7 gene locus.
  • High-molecular weight DNAs from cancer cells were cleaved using restriction endonuclease EcoRI, separated electrophoretically, transferred to nylon membrane, and probed with the 1.7 kilobase EEZ7 ORF probe (S ⁇ Q ID NO: 3).
  • the DNAs applied to each lane (10 micrograms per lane) were obtained from the following cells: lane 1, cell line MB436S; lane 2, normal placental cells obtained from a first healthy individual.; lane 3, cell line MB231 ; lane 4, cell line MB361 ; lane 5. cell line T ⁇ 8; and lane 6, cell line TE3.
  • the DNA applied to lane 7 was isolated from normal placental cells obtained from a second healthy individual.
  • Figure 4 comprises Figures 4A and 4B.
  • Figure 4A is a diagram which depicts truncated EEZ7 transcripts observed in cancer cells, as described herein.
  • the normal exon/intron structure is indicated on the top line of the diagram, and was determined by sequencing of normal (i.e. non-cancerous) brain, prostate and esophagus cDNAs and by sequencing EEZ7 gene in BAC. Boxes represent exons; the hatched areas represent the open reading frame (1788 base pairs; S ⁇ Q ID NO: 3). Horizontal lines represent introns. and closed circles represent point mutations which were observed, as described herein.
  • the boxed notation "LZ” represents the approximate location of the leucine-zipper motif described herein.
  • FS represents the approximate position of a frame-shift described herein. Aberrant transcripts observed in tumors are depicted by bold lines on the lines below the top line in the diagram.
  • Figure 4B is the putative amino acid sequence (S ⁇ Q ID NO: 6) encoded by the frame-shifted EEZ7 transcript having a molecular weight of about 8.6 kilodaltons. Amino acid residues encoded by the frame-shifted portion of the transcript are underlined.
  • Figure 5 is a series of nucleotide and amino acid sequences.
  • Figure 5A comprises Figures 5Ai-5Avi. and lists the nucleotide sequence (SEQ ID NO: 1) of a portion of the human genome comprising the EEZ7 gene.
  • Figure 5B comprises Figures 5Bi-5Biv, and lists the nucleotide sequence (S ⁇ Q ID NO: 2) of a cDNA which reflects the nucleotide sequence of the full-length mRNA transcript of wild type EEZ7.
  • Figure 5C lists the nucleotide sequence (S ⁇ Q ID NO: 9) of a cDNA which reflects the nucleotide sequence of the ORF region of a truncated ( ⁇ 16T8) EEZ7 mRNA transcribed by tumors cells.
  • Figure 5D lists the nucleotide sequence (S ⁇ Q ID NO: 10) of a cDNA which reflects the nucleotide sequence of the ORF region of a truncated ( ⁇ 264162) EEZ7 mRNA transcribed by tumors cells.
  • Figure 5 ⁇ comprises Figures 5Ei and 5Eii.
  • Figure 5F comprises Figures 5Fi and 5Fii, and lists the nucleotide sequence (S ⁇ Q ID NO: 12) of a cDNA which reflects the nucleotide sequence of the ORF region of a truncated (D14) EEZ7 mRNA transcribed by tumors cells.
  • Figure 5G comprises Figures 5Gi and 5Gii, and lists the nucleotide sequence (S ⁇ Q ID NO: 13) of a cDNA which reflects the nucleotide sequence of the ORF region of a truncated (G3611) EEZ7 mRNA transcribed by tumors cells.
  • Figure 5H comprises Figures 5Hi and 5Hii, and lists the nucleotide sequence (S ⁇ Q ID NO: 14) of a cDNA which reflects the nucleotide sequence of the ORF region of a truncated (G3612) EEZ7 mRNA transcribed by tumors cells.
  • Figure 51 comprises Figures 5Ii and 5Iii, and lists the nucleotide sequence (S ⁇ Q ID NO: 3) of a cDNA which reflects the nucleotide sequence of the ORF region of wild type EEZ7 mRNA.
  • Figure 5J comprises Figures 5Ji-5Jv, and lists the amino acid sequence (S ⁇ Q ID NO: 4) of full- length, human wild type Fezl protein.
  • Figure 5K lists the amino acid sequence (S ⁇ Q ID NO: 15) of a truncated ( ⁇ 16T8) Fezl protein expressed by tumors cells.
  • Figure 5L comprises Figures 5Li and 5Lii, and lists the amino acid sequence (SEQ ID NO: 16) of a truncated (E264162) Fezl protein expressed by tumors cells.
  • Figure 5M comprises Figures 5Mi-5Miv, and lists the amino acid sequence (SEQ ID NO: 17) of a truncated (T8D145M4) Fezl protein expressed by tumors cells.
  • Figure 5N comprises Figures 5Ni-5Niv. and lists the amino acid sequence (SEQ ID NO: 18) of a truncated (D14) Fezl protein expressed by tumors cells.
  • Figure 50 comprises Figures 5Oi-5Ov.
  • Figure 5P comprises Figures 5Pi- 5Pv, and lists the amino acid sequence (SEQ ID NO: 20) of a truncated (G3612) Fezl protein expressed by tumors cells.
  • Figure 5Q lists the nucleotide sequence (SEQ ID NO: 21) of the F37 probe described herein.
  • Figure 6 is an image of an immunoblot of proteins isolated from MCF7 cell line clones which had been transfected with pTet-OffTM vector alone ("control") or with the vector having at least the coding portion of the EEZ7 gene operably linked with the promoter thereof (clones 118, 54, 18, and 15). Proteins were isolated from cells which had been maintained in the presence ("+”) or absence ("-") of tetracycline.
  • Figure 7 comprising Figures 7A (clone 15), 7B (clone 54), 7C (clone 18), and 7D (clone 118), is a quartet of graphs which indicate the time dependence of the ratio of transfected MCF7 clone cell number to control cell number for cells maintained in tetracycline-free medium containing 10% (o), 5% (•), 2.5% (a), 1% ( ⁇ ), or 0.5% (A) (v/v) fetal bovine serum.
  • Figure 8 is a pair of graphs which indicate the ratios of the number of transfected MCF7 clone cells which were in the G2 cell cycle stage, relative to the number in the M stage ( Figure 8A; i.e.. G2/M) or the ratio of the number of cells in the S phase, relative to the number in the Gl stage ( Figure 8B; i.e.. S/Gl).
  • solid lines correspond to clone 18
  • broken lines correspond to clone 54.
  • Filled circles correspond to ratios in the of presence tetracycline (i.e. non-expression of FEZl), and open circles correspond to ratios in the absence of tetracycline (i.e. expression of FEZl).
  • Figure 9 is a pair of graphs which indicate the temporal dependence of tumor volume in nude mice into which about 5 x 10 6 ( Figure 9A) or about 2 x 10 7 ( Figure 9B) MCF7 cells transfected with vector alone (o). transfected MCF7 clone 15 cells (•), transfected MCF7 clone 18 cells ( ⁇ ),
  • R ⁇ CTIF1 ⁇ D SHEET (RULE 91) transfected MCF7 clone 56 cells (G), or transfected MCF7 clone 1 18 cells (0) were implanted.
  • Figure 10 comprising Figures 10A-10F, lists the nucleotide sequence (SEQ ID NO: 60) of pQBI-AdCMV5-IRES-GFP.
  • Figure 11 comprising Figures 11A, 1 IB, and 11C. is a trio of images of the results of an in vitro binding assay demonstrating binding between 35 S- methionine-labeled EFl- ⁇ and Fezl protein.
  • Figure 12 is an image of the results of an in vitro binding assay demonstrating binding between 35 S-methionine-labeled EFl- ⁇ (N) and Fezl protein and between EF 1 - ⁇ (N) and a truncated Fezl protein.
  • Figure 13 is an image of the results of an in vitro binding assay demonstrating dimerization of Fezl protein and dimerization of truncated Fezl protein.
  • Figure 14 comprising Figures 14A, 14B, 14C, and 14D, is a series of four images which depict the results of immunoblotting experiments involving HeLaS3 cells which were co-transfected with a vector encoding a V5/Fezl fusion protein and a vector encoding an EXP/EF 1 - ⁇ fusion protein.
  • Figure 15. comprising Figures 15A and 15B, is a pair of images of the results of immunoblotting experiments.
  • Figure 16, comprising Figures 16A and 16B. is a pair of images of the results of immunoblotting experiments in synchronized, transfected MCF7 cells, using an antibody which binds specifically with Fezl ("Fezl”) and an antibody which binds specifically with actin ("actin"). Numbers above the columns indicate the elapsed time following aphidicolin treatment.
  • Fezl Fezl
  • actin actin
  • the proteins immunoblotted in the experiments corresponding to Figure 16A were obtained from transfected MCF7 cells which were maintained in the presence of 10% (v/v) FBS, and the proteins immunoblotted in the experiments corresponding to Figure 16B were obtained from transfected MCF7 cells which were maintained in the absence of FBS.
  • Figure 17 is an image of the results of an immunoblotting experiment involving proteins extracted from cell cycle-synchronized fetal kidney 293 cells.
  • Figure 18 is an image of the results of an SDS-PAGE separation of the proteins obtained from the cells corresponding to Figure 16A. The cells corresponding to lanes 1, 2, 3, and 4 in Figure 18 correspond to lanes designated 0, 1.5, 5, and 9 in Figure 16A.
  • Figure 19 is an image of the results of an immunoblotting experiment in which the cell lysates used in the experiments corresponding to Figure 16A were contacted with alkaline phosphatase (lane 1), ⁇ -glycerophosphate (lane 2), or a control.
  • Figure 20 is an image of SDS-PAGE separated Fezl proteins obtained from synchronized, transfected MCF7 cells and immunoblotted with either an anti- Fezl polyclonal antibody (lanes 6-10) or an anti-phosphoserine antibody (lanes 1-5) at increasing times from 0 (lanes 1 and 6) to 8 hours (lanes 5 and 10) following cessation of cell cycle inhibition.
  • Figure 21 is an image of the results of an experiment in which cytoplasmic (“Cl “ and “C2”) and nuclear (“N”) protein extracts obtained from 293 cells were immunoblotted using a polyclonal anti-Fez 1 antibody (“Fezl ”) or an anti- tubulin antibody (“tubulin”).
  • Figure 22 is an image of the results of an experiment in which Fezl protein which Fezl protein "Fezl " was detected using a polyclonal antibody in extracts obtained from centrifugation- sedimented cell structures in synchronized cells which had been incubated with paclitaxel ("Tax”) or with colchicine ("Col”).
  • Figure 23 is a graph which indicates the effect of Fezl protein on inhibition of tubulin polymerization in the presence of MAP2 protein.
  • Reaction mixtures contained, in addition to reaction buffer: nothing (open circle); tubulin (open diamond); tubulin and MAP2 (open square); tubulin, MAP2, and GST (diamond enclosing cross); tubulin, MAP2, and GST-fused Fezl (filled circle); tubulin, MAP2, GST-fused mutated (29 Ser Pro) Fezl (filled square); tubulin, MAP2.
  • PKA protein kinase A, a 3' :5'-monophosphate-dependent protein kinase.
  • the present invention is based on the discovery, isolation, and sequencing of FEZl, a tumor suppressor gene located at human chromosome location 8p22. It was observed that decreased, or no, expression of FEZl could be detected in a variety of cancer cells obtained from cancer cell lines and cancer tissue samples taken from human patients. Cancer types in which abnormal (i.e. decreased or no) expression of FEZl has been detected include, but are not limited to, epithelial cancers, cancers of the digestive system, esophageal cancers, gastric cancers, colon cancers, prostate cancers, breast cancers, hematopoietic cancers, lung cancers, melanomas, and cervical cancers, as described herein.
  • FEZl FEZl protein kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase, and the like.
  • post-translational phosphorylation and de-phosphorylation of Fezl protein can modulate the effect that Fezl protein has on these physiological processes.
  • a “polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid.
  • a polynucleotide can be either a single-stranded or a double-stranded nucleic acid.
  • an "isolated" polynucleotide is one which refers to a nucleic acid segment or fragment which is separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which is not adjacent to the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which are substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • an "isolated" protein or antibody is one which is separate from one or more other components which naturally accompany it in its naturally occurring state.
  • an isolated protein can be prepared by separating a protein from at least one other protein which naturally accompanies it. Further by way of example, an isolated protein can be prepared by synthesizing the protein in the absence of at least one other protein which naturally accompanies it.
  • a "substantially purified" polynucleotide, protein, or antibody is one which is separate from at least most of the components which naturally accompany it in its naturally occurring state, and preferably from at least 75%. 80%. 90%, or even 95% of those components, as assessed on a per- weight basis or a per-mole basis.
  • homologous refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions, e.g..
  • “Completely homologous” means having 100% homology. “Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is anti-parallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is anti-parallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if. when the two regions are arranged in an anti-parallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an anti-parallel fashion, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion, in which event, the two portions are described as being “completely complementary.”
  • substantially complementary means having at least 70%, 75%. 80%. 85%, 90%. 95%, 97%, 98%, or even at least 99% complementarity.
  • a first polynucleotide "anneals" with a second polynucleotide if the nucleotide residues of at least one region of each of the two polynucleotides participate in base pairing when the two regions are arranged in an anti-parallel fashion in an appropriate solution.
  • solutions include, e.g. standard saline citrate (SSC) buffer.
  • a first polynucleotide anneals "with high stringency" with a second polynucleotide if the two polynucleotides anneal under conditions whereby only oligonucleotides which are at least about 75%, and preferably at least about 90% or at least about 95%. complementary anneal with one another.
  • the stringency of conditions used to anneal two polynucleotides is a function of. among other factors, temperature, ionic strength of the annealing medium, the incubation period, the length of the polynucleotides. the G-C content of the polynucleotides, and the expected degree of non-homology between the two polynucleotides, if known.
  • high stringency hybridization conditions include hybridizing conditions that ( 1 ) employ low ionic strength and high temperature for washing, for example. 0.015 molar NaCl. 1.5 millimolar sodium citrate, and 0.1% (w/v) sodium dodecyl sulfate (SDS) at 50°C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (v/v) formamide. 0.1% (w/v) bovine serum albumin.
  • a “functional” or “operative” protein is a protein in a form which exhibits at least one biological activity by which it is characterized in its naturally occurring state.
  • a “functional” or “operative” gene is a gene which, when present in an environment comprising functional gene expression proteins (e.g. the interior of a human cell or an in vitro gene expression mixture of a type described in the art), is expressed to yield the gene product encoded or specified by the gene.
  • a first polynucleotide is "specified” by a second polynucleotide if the first polynucleotide is either homologous with or complementary to a transcript polynucleotide generated either by transcription or by reverse transcription of at least a portion of the second polynucleotide.
  • the first polynucleotide can be homologous with or complementary to the transcript polynucleotide either before or after the transcript polynucleotide has been acted upon by eukaryotic mRNA splicing components.
  • a "portion" or "region” of a polynucleotide means at least two consecutive nucleotide residues of the polynucleotide. and preferably at least 10, 11.
  • a first portion of a polynucleotide is "adjacent" a second portion of the same polynucleotide if the nucleotide sequences of the first and second portions are directly attached to one another, having no intervening nucleotides.
  • the pentanucleotide 5'-AAAAA-3' is adjacent the trinucleotide 5'-TTT-3' when the two are connected thus: 5'-AAAAATTT-3' or 5'-TTTAAAAA-3'. but not when the two are connected thus: 5'-AAAAACTTT-3'.
  • a first portion of a polynucleotide "flanks" a second portion of the same polynucleotide if the two portions are adjacent one another or if the two portions are separated by no more than about 1000, 999, 998 900. 899. 898 750, 749. 748, ..., 500, 499. 498, ..., 250, 249, 248. ..., and preferably no more than about 100 nucleotide residues.
  • two polynucleotides as "operably linked” is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other.
  • a promoter operably linked with the coding region of a gene is able to promote transcription of the coding region.
  • promoter means a nucleic acid sequence which is required for expression of a gene product operably linked with the promoter sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • a “constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell under most or all physiological conditions of the cell.
  • An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • substantially absence of expression of a gene means that the level of expression of the gene is undetectable or is at least greatly reduced (e.g. 100-fold or 1000-fold or more) relative to expression of the gene in its naturally occurring state.
  • an "expression vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell, such that a gene product encoded by or specified by the isolated nucleic acid is generated in the cell.
  • Numerous expression vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • Expression vectors generally either comprise a promoter operably linked with a portion of the isolated nucleic acid which encodes or specifies a gene product, or are capable of inserting the isolated nucleic acid into a cellular nucleic acid wherein the portion is operably linked with a cellular promoter.
  • an exogenous polynucleotide in an organism is one which is not present in a naturally-occurring form of the organism in the same form as the polynucleotide.
  • an exogenous polynucleotide can be one which comprises a nucleotide sequence which the genome of the organism does not comprise, or it can be one which comprises a portion of the organism's genome in a form (e.g. a plasmid or an artificial chromosome) which is not present in a naturally-occurring form of the organism.
  • an “analog” of a gene is one is substantially homologous with the gene and which encodes or specifies a gene product having a biological activity which is substantially the same as a biological activity exhibited by the gene product encoded or specified by the gene.
  • a "FEZl -associated polynucleotide” means a polynucleotide which comprises a portion which is substantially homologous with or substantially complementary to at least about 20. 21, 22, .... 30, 31. 32, ..., 40. 41. 42, .... or 50 or more consecutive nucleotide residues of either a human EEZ7 gene or a spliced mRNA specified by a human EEZ7 gene.
  • a "EEZ -transcript-associated polynucleotide” means a polynucleotide which comprises a portion which is substantially homologous with or substantially complementary to at least about 20, 21, 22, ..., 30, 31, 32, ..., 40. 41, 42, .... or 50 or more consecutive nucleotide residues of either a spliced or non-spliced mRNA specified by a human EEZ7 gene.
  • Contigs of a genomic region are a collection of oligonucleotides, usually contained in a yeast, bacterial, or phage vector, which together include all or substantially all (i.e. >95%, and preferably >99%) of the sequence of the genomic region.
  • An "exon boundary polynucleotide probe” is a polynucleotide which is complementary to or homologous with at least five nucleotide residues of an exon of a EEZ7 gene which are adjacent to an intron of that gene.
  • protein-ligand pair refers to a protein and another molecule, wherein the protein specifically binds with the other molecule.
  • protein- ligand pairs include an antibody and its corresponding epitope and an avidin protein, such as streptavidin, and biotin.
  • a protein or polynucleotide is "detectably labeled” if the protein or polynucleotide comprises or is linked with a composition of matter which can be detected after contacting the protein or polynucleotide with another protein or polynucleotide.
  • detectable labeling proteins and polynucleotides including, for example, surfaces with which such compounds are linked, radionuclides incorporated into such proteins, chromophores and fluorophores which are linked with such compounds, and the like.
  • a “gene chip” is a manufacture comprising a surface having an ordered array of polynucleotides attached thereto, either permanently or reversibly.
  • the ordered array can comprise four sections, wherein one of four polynucleotides is attached to the surface in each section, and wherein the four polynucleotides have nucleotides sequences which are identical with the exception of one nucleotide residue (e.g. 5'-AACCAAAAAAA-3'; 5'-AACCAAAAAAAT-3'; 5'-AACCAAAN AC-3'; and 5'-AACCAAAAAAG-3').
  • An “inducer of cell proliferation” is a composition of matter which, when contacted with a cell, causes the cell to grow, divide, or replicate at a rate greater than the corresponding rate in the absence of the composition.
  • Cell proliferation is "retarded” if the rate of cell proliferation is reduced.
  • tissue or cell refers to whether the cell or one or more cells within the tissue have accumulated enough genomic mutations that they either presently exhibit one or more characteristics of tumor cells or tissue (e.g. uncontrolled cell proliferation or metastasis) or, will, without further genomic damage, exhibit one or more characteristics of tumor cells or tissue upon incubation or maintenance of the cell.
  • a "phenotypically abnormal" portion of a tissue is one which comprises cells which have one or more characteristics of cancer cells of the tissue type such as. for example, abnormal morphology or abnormal growth or proliferation rate.
  • a "phenotypically normal" portion of a tissue is one which does not appear to be phenotypically abnormal.
  • a “candidate anticancer compound” is a compound which has exhibited potential anti-cancer activity in a relevant assay or a compound which has substantial structural similarity to such a compound. Methods of identifying a compound which exhibits potential anti-cancer activity and methods of designing structurally similar compounds are well known in the art.
  • pharmaceutically acceptable carrier means a chemical composition with which one or more active ingredients can be combined and which, following the combination, can be used to administer one or more active ingredients to a subject.
  • physiologically acceptable ester or salt means an ester or salt form of an active ingredient which is compatible with any other ingredients of the pharmaceutical composition and which is not deleterious to the subject to which the composition is to be administered.
  • An "instructional material” means a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of an isolated polynucleotide, an isolated protein, or a pharmaceutical composition of the invention for performing one or more of the methods of the invention.
  • the instructional material may, for example, describe how to use one of these compositions to perform a diagnostic method of the invention, a therapeutic method of the invention, or a screening assay of the invention, or, for example, an appropriate dose of a pharmaceutical composition of the invention.
  • a “tubulin hyperpolymerization disorder” is a disorder which is associated with a greater extent or rate of tubulin polymerization in a cell or animal afflicted with the disorder than in a cell or animal which is not afflicted with the disorder.
  • a “tubulin hypopolymerization disorder” is a disorder which is associated with a lesser extent or rate of tubulin polymerization in a cell or animal afflicted with the disorder than in a cell or animal which is not afflicted with the disorder.
  • Description Being a tumor suppressor gene, EEZ7 is intimately involved in control of the cancerous or non-cancerous phenotype of a cell which normally expresses it. Characteristics of tumor cells which normally express EEZ7 include abnormal cell proliferation, abnormal cell growth, and abnormal differentiation of cells.
  • FEZl limits cell proliferation. While not wishing to be bound by any particular theory of operation, it is thought that a leucine-zipper region described herein within the putative structure of Fezl protein is involved in binding between Fezl and one or more regions of a physiological polynucleotide (e.g. genomic DNA), whereby expression (i.e. transcription or translation) of the polynucleotide is inhibited or prohibited. Binding between Fezl and one or more regions on the human genome can inhibit transcription of one or more genes located nearby on the genome, and is contemplated as a potential mechanism of action for EEZ7 regulation of cell proliferation.
  • a physiological polynucleotide e.g. genomic DNA
  • the nucleotide sequence (S ⁇ Q ID NO: 1) of a portion of the human genome encoding wild type EEZ7 is shown in Figure 5A.
  • the nucleotide sequence (S ⁇ Q ID NO: 2) of cDNA generated using full-length mRNA transcribed from wild type EEZ7 is shown in Figure 5B.
  • the nucleotide sequence (S ⁇ Q ID NO: 3) of the open reading frame (ORF) of wild type EEZ7 is shown in Figure 51.
  • the putative amino acid sequence (S ⁇ Q ID NO: 4) of wild type Fezl protein is shown in Figure 5J.
  • Nucleotide sequences (S ⁇ Q ID NOs: 9-14) of cDNAs generated using truncated EEZ7 mRNA species and amino acid sequences (S ⁇ Q ID NOs: 15-20) of corresponding truncated Fezl proteins are shown in Figures 5C to 5H and in Figures 5K to 5P, respectively.
  • the invention includes an isolated polynucleotide which anneals with high stringency with at least twenty consecutive nucleotide residues of at least one strand of the human EEZ7 gene, such as a human gene having the sequence S ⁇ Q ID NO: 1.
  • the isolated polynucleotide of the invention anneals with high stringency with at least 20. 21. 22, .... 30, 31, 32, ..., 50, 51, 52, ..., 75, 76, 77, ..., or 100 consecutive nucleotide residues of at least one strand of the human EEZ7 gene, or is substantially complementary with those residues.
  • the isolated polynucleotide of the invention have a length not greater than about 200, 199.
  • the isolated polynucleotide of the invention preferably has a sequence that is substantially homologous with at least 20, 21. 22. ..., 30. 31 , 32, ..., 40, 41, 42, ..., or 50 consecutive nucleotide residues of at least one strand of the human EEZ7 gene. More preferably, the isolated polynucleotide of has a sequence completely homologous with at least 20, 21, 22, ..., 30, 31. 32.
  • the isolated polynucleotide of the invention can be selected to be homologous with either the coding strand or the non-coding strand of FEZl.
  • the isolated polynucleotide can comprise both a first portion that is homologous with one strand of FEZl and a second portion that is homologous with the other strand, such an isolated polynucleotide that is capable of forming a hairpin-type structure when the first portion thereof anneals with the second.
  • the skilled artisan will be able, in light of the present disclosure, to decide whether the isolated polynucleotide should comprise a portion homologous with the coding strand of FEZl. a portion homologous with the non-coding strand, or both.
  • the degree of homology between the isolated polynucleotide and the at least one strand of human EEZ7 can be more or less critical in various embodiments described herein.
  • the necessary degree of homology between the isolated polynucleotide and the at least one strand of FEZl is dependent on the length of the polynucleotide.
  • the degree of complementarity necessary to anneal the polynucleotide with another polynucleotide with high stringency decreases. Numerous methods, algorithms, computer programs, and the like are known whereby the skilled artisan can predict the stringency of binding between two polynucleotides (e.g.
  • the determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.
  • NBLAST nucleotide searches can be performed with the NBLAST program (designated
  • BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes. Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).
  • PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern.
  • BLAST Altschul et al.
  • Gapped BLAST PSI-Blast
  • PHI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • the isolated polynucleotide of the invention is to be used to express all or a portion of a human Fezl protein, either in vitro or in vivo, it is important that (i) the homology of the isolated polynucleotide with the human EEZ7 gene (e.g.
  • S ⁇ Q ID NO: 1) is such that the amino acid sequence encoded by the isolated polynucleotide is identical to the corresponding region of FEZl, (ii) the differences between the sequence of the isolated polynucleotide and the corresponding region of EEZ7 not result in differences in the encoded amino acid sequence (i.e. any sequence difference in a coding region merely substitutes a codon encoding an amino acid in place of another codon encoding the same amino acid), or (iii) any differences in the encoded amino acid sequence between the isolated polynucleotide and the corresponding region of FEZl results only in one or more conservative amino acid substitutions, as described in greater detail elsewhere herein.
  • the following Human Codon Table can be used to seiect or identify alternate codons which encode the same amino acid.
  • nucleotide residue changes into a polynucleotide such as the isolated polynucleotide of the invention, or into a Fezl protein or a portion thereof
  • a variety of well-known techniques can be used, such as site-specific mutagenesis.
  • Site-specific mutagenesis allows production of mutants through the use of specific oligonucleotides which encode the sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complementarity to form a stable duplex on both sides of the nucleotide sequence to be altered (e.g. a codon).
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • This technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as Ml 3 phage. Such vectors are commercially available, and their use is well known in the art.
  • Double stranded plasmids are also routinely employed in site-directed mutagenesis protocols, to eliminate the need to transfer the gene of interest from a plasmid to a phage vector.
  • Site-directed mutagenesis is performed by first obtaining a single-stranded vector or dissociating the two strands of a double stranded vector which includes within its sequence a DNA sequence which comprises the desired site of mutagenesis.
  • the oligonucleotide primer described above is annealed with the single-stranded vector, and subjected to DNA polymerization, in order to generate a mutation-bearing strand.
  • a heteroduplex is formed between the mutation-bearing strand and either the original non-mutated strand of the double-stranded vector or an added or synthesized strand which is substantially complementary to the mutation-bearing strand. This heteroduplex is then used to transform appropriate cells, such as E.
  • sequence variants of the isolated polynucleotide of the invention using site-directed mutagenesis is provided merely as an example of a method of producing potentially such variants, and is not intended to be limiting, as there are other well-known methods for producing such variants.
  • recombinant vectors comprising or encoding the desired isolated polynucleotide can be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • the isolated polynucleotide of the invention can be single stranded or double-stranded, it being understood that a single-stranded form is the form referred to herein when annealing of the isolated polynucleotide of the invention with another nucleic acid is described.
  • the isolated polynucleotide of the invention can be substantially any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides. and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate. siloxane, carbonate, carboxymethyl ester, acetamidate. carbamate, thioether. bridged phosphoramidate, bridged methylene phosphonate. bridged phosphoramidate. bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate. methylphosphonate, phosphorodithioate. bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).
  • the isolated polynucleotide of the invention is preferably in a substantially purified form. It is not intended that the present invention be limited by the nature of the nucleic acid employed.
  • the isolated polynucleotide of the invention can be an isolated, naturally occurring nucleic acid or it can be a synthetic nucleic acid.
  • the isolated, naturally occurring nucleic acid can obtained be from a viral, bacterial. animal, human, or plant source.
  • the polynucleotide can be DNA or RNA.
  • nucleic acid can be isolated, synthesized, or assembled as part of a virus or other macromolecule. See, e.g., Fasbender et al, 1996. J. Biol. Chem. 272:6479-89 (polylysine condensation of DNA in the form of adenovirus).
  • Nucleic acids useful in the present invention include, by way of example and not limitation, oligonucleotides and polynucleotides such as antisense DNAs and/or RNAs; ribozymes; DNA for gene therapy; viral fragments including viral DNA and/or RNA: DNA and or RNA chimeras; mRNA; plasmids; cosmids: genomic DNA; cDNA; gene fragments; various structural forms of DNA including single-stranded DNA, double stranded DNA, supercoiled DNA and/or triple-helical DNA; Z-DNA; and the like.
  • the nucleic acids can be prepared by any conventional means typically used to prepare nucleic acids in large quantity.
  • DNAs and RNAs can be chemically synthesized using commercially available reagents and synthesizers by methods that are well-known in the art (see, e.g., Gait, 1985, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, England)).
  • RNAs can be produce in high yield via in vitro transcription using plasmids such as SP65 (Promega Corporation. Madison, WI).
  • nucleic acids having modified internucleoside linkages can be preferred.
  • Nucleic acids containing modified internucleoside linkages can also be synthesized using reagents and methods that are well known in the art. For example, methods for synthesizing nucleic acids containing phosphonate, phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal. thioformacetal.
  • Stability of the isolated polynucleotide of the invention can also be enhanced by treating on or both ends of the polynucleotide (if it is linear) with at least one agent which nucleolytically blocks the end.
  • agents are known in the art (e.g. agents described in Oligonucleotides as Therapeutic Agents, 1997, John Wiley & Sons. New York).
  • the isolated polynucleotide can be purified by any suitable means, such as are well known in the art.
  • the isolated polynucleotide can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography, or gel electrophoresis.
  • the method of purification will depend in part on the size and type of the nucleic acid to be purified and on the characteristics of any molecules, structure, or organisms with which it can be associated.
  • the isolated polynucleotide of the invention can comprise nucleotide residues other than the five naturally occurring bases, adenine, guanine. thymine. cytosine. and uracil.
  • the isolated polynucleotide of the invention is detectably labeled.
  • Any known method of labeling a nucleic acid can be used to label the polynucleotide.
  • well known methods of detectably labeling a polynucleotide include incorporation of a radionuclide into the polynucleotide, linking the polynucleotide to a surface, such as a latex bead or a nylon membrane, linking a protein such as an enzyme to the polynucleotide, linking one of a protein-ligand pair (e.g. an avidin-biotin pair or an antibody-antigen pair) to the polynucleotide.
  • a protein-ligand pair e.g. an avidin-biotin pair or an antibody-antigen pair
  • the isolated polynucleotide is reversibly linked with both a fluorophore and a molecule capable of quenching the fluorescence of the fluorophore. whereby if either the fluorophore or the quenching molecule is dissociated from the isolated polynucleotide.
  • the isolated polynucleotide of the invention has numerous uses. For example, such an isolated polynucleotide can be detectably labeled and used as a probe to detect the presence of a different polynucleotide having a sequence comprising a portion to which it anneals (e.g.
  • Such a probe can be used, for example, to detect or to quantify expression of EEZ7 in a cell or tissue of a human. It is understood that numerous methods of using a polynucleotide probe for detection and quantification of nucleic acids with which the probe anneals are known in the art (e.g. Sambrook et al.. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. New York: Ausubel et al., 1992. Current Protocols in Molecular Biology. John Wiley & Sons, New York; Gerhardt et al..
  • the probe When the probe is used for detection or quantification of a nucleic acid encoding all or a portion of FEZl, it is preferably detectably labeled.
  • the isolated polynucleotide of the invention can similarly be used to detect the presence of a non-human analog of the human EEZ7 gene in a polynucleotide obtained or derived from a non-human source (e.g. a library of genomic fragments obtained from, or a library of cDNAs derived from mRNAs of, an animal such as a mammal).
  • nucleotide sequence of FEZl will be more highly conserved among organisms than less critical genes.
  • the isolated polynucleotide of the invention is useful not only for isolation and identification of primate and other mammalian EEZ7 analogs, but also for isolating and identifying other vertebrate, other eukaryotic, and possibly any FEZl analog.
  • a non-human analog of FEZl is to be isolated or identified
  • a plurality of isolated polynucleotides of the invention are used, each polynucleotide being complementary to a different portion of human FEZl.
  • at least one isolated polynucleotide of the invention is complementary to a portion of human EEZ7 which can be expected to be particularly conserved, such as the portion which encodes the leucine -zipper region of Fezl protein.
  • a manufacture comprising a plurality of isolated polynucleotide probes of the invention fixed in an ordered array on a surface.
  • Such manufactures are colloquially known as 'gene chips.
  • Each of the plurality of probes anneals with high stringency with a portion of the human EEZ7 gene.
  • nucleic acids which comprise different nucleotide residues at that position within the EEZ7 gene can be differentiated.
  • missense and deletion mutations in the EEZ7 sequence can be detected.
  • the nucleotide sequence of all. or any portion, of the EEZ7 gene can be determined.
  • the wild type human EEZ7 gene sequence which is used is S ⁇ Q ID NO: 1.
  • An analogous ordered array can be designed to detect mRNA sequence alterations, preferably using S ⁇ Q ID NO: 2 or S ⁇ Q ID NO: 3 as the wild type human EEZ7 m-RNA sequence. Manufactures of this type are analogous to the GeneChipTM devices made by Affymetrix. Inc. (Santa Clara.
  • CA which comprise pluralities of primers which bind with high stringency to, for example, portions of the human p53 gene or to portions of the HIV-1 protease or reverse transcriptase genes.
  • Methods for making and using such manufactures have been described elsewhere, and need only be modified by the skilled artisan to include the EEZ7 gene sequences described in the present disclosure (Wallraff et al.. February 1997. Che tech 22-23; Lockhart et al., 1996, Nature Biotechnol. 14:1675-1680; Pease et al. 1994. Proc. Natl. Acad. Sci. USA 91 :5022- 5026: Fodor et al.. 1993. Nature 364:555-556).
  • One or more isolated polynucleotides of the invention can also be used as primers for replication or amplification of all or a portion of a nucleic acid comprising all or pan of a human EEZ7 gene or a non-human EEZ7 analog.
  • the nucleic acid may. for example, be either strand of a human genome, a human chromosome, a fragment of a human genome, or all or a portion of a non-human genome, or it can be an mRNA generated by transcription of a human EEZ7 gene or a non-human analog thereof or either strand of a cDNA generated using such an mRNA.
  • the skilled artisan can replicate or amplify substantially any nucleic acid comprising a portion homologous with or complementary to all, or a portion, of a human EEZ7 gene, such as that having the nucleotide sequence S ⁇ Q ID NO: 1.
  • Methods of DNA transcription, RNA reverse transcription. DNA replication, polymerase chain reaction (PCR), and the like are well known and not described beyond citation to the following standard references (Sambrook et al.. 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1992, Current Protocols in Molecular Biology.
  • the polynucleotides should be selected such that one polynucleotide anneals with one strand with high stringency near one end of the region to be amplified and the other polynucleotide anneals with the other strand with high stringency near the other end of the region to be amplified, as is well known in PCR methods.
  • the nucleic acid to be amplified is an mRNA or other RNA molecule, then a cDNA complementary to the mRNA must be made prior to performing a PCR reaction.
  • Substantially any region of the human EEZ7 gene, or of a non-human analog thereof, can be amplified using one or more isolated polynucleotides of the invention.
  • polynucleotides which anneal with high stringency with at least 20, 21, 22, .... 30, 31, 32 40. 41. 42 or 50 nucleotide residues near opposite ends and on opposite strands of the human EEZ7 gene are used to amplify the entire human EEZ7 gene, or a non-human analog thereof, from one or more portions of a human or non-human genome.
  • one or more pairs of isolated polynucleotide primers are selected, each of which pairs of primers comprises a first primer which anneals with high stringency with an intronic portion which flanks the 5'- or 3 '-end of an exon on the coding strand of a nucleic acid encoding the exon and a second primer which anneals with high stringency with an intronic portion which flanks the 3'- or 5'- end. respectively, of the same exon on the non-coding strand of the nucleic acid.
  • each of the two primers of each pair is adjacent the designated end of the exon.
  • amplification of a nucleic acid encoding at least one exon of the human EEZ7 gene, or a non-human analog thereof, using one or more pairs of primers results in amplification of one or more exon sequences of the gene or analog, optionally not including any intronic sequence. It is understood that amplification of both an exon sequence and the intronic sequences which flank it can be more informative than amplification of exon sequences alone, since sequence alterations which appear in an intron but nonetheless affect the amino acid sequence of the encoded protein (e.g. mutations which affect mRNA splicing) can be revealed.
  • pairs of isolated polynucleotide primers of the invention are selected such that amplification of the wild type human genomic EEZ7 region (e.g. S ⁇ Q ID NO: 1), the corresponding wild type mRNA, or a cDNA generated from wild type human EEZ7 mRNA using these pairs of primers yields a mixture of amplification products having determined lengths. Fractionation of these amplification products by size (e.g. by gel electrophoresis or by chromatography) will yield a characteristic pattern for the wild type sequence. Amplification of the same nucleic acid obtained from an individual having a mutation which affects the length or presence of any of the amplification products will yield a different pattern than the wild type pattern, and the presence of the mutation in the individual can thus be identified.
  • the wild type human genomic EEZ7 region e.g. S ⁇ Q ID NO: 1
  • a cDNA generated from wild type human EEZ7 mRNA using these pairs of primers yields a mixture of amplification products having determined lengths. Fr
  • pairs of isolated polynucleotide primers of the invention are selected in order to amplify regions of a nucleic acid encoding human Fezl protein, or a non-human analog thereof, which are known to be altered (i.e. wherein a deletion or missense mutation are known to occur) in tumor cells.
  • regions are described herein in Example 1 , and primers useful for amplifying these regions are included in the invention. Identification of the presence of such alterations is an indication that the cell or tissue from which the nucleic acid was obtained is cancerous.
  • primers useful in this embodiment include, for example, primer pairs G12 and G13.
  • the isolated polynucleotide of the invention can also be used as an antisense oligonucleotide (ASO) to inhibit expression of a human EEZ7 gene or a non- human analog thereof.
  • ASO antisense oligonucleotide
  • an ASO can be complementary to either the coding or non-coding strand of a gene.
  • ASOs are used by delivering the ASO to the interior of a cell, and preferably to the interior of the nucleus of a cell, whereby the ASO is enabled to interact with one or more nucleic acids which encode a protein.
  • an isolated polynucleotide of the invention is used as an ASO. it binds with high stringency with at least 20, 21, 22, .... 30. 31.
  • the isolated polynucleotide ASO of the invention is preferably substantially homologous. and more preferably completely homologous with at least 20, 21, 22 30. 1, 32. ...,
  • the isolated polynucleotide ASO is preferably substantially or completely homologous with the translation start site, the transcription start site, an exon-intron boundary for splicing immature mRNA, or a coding sequence of the human FEZ7 gene.
  • Other prefened ASO are complementary to or homologous with and approximately about as long as the EEZ7 ORF (S ⁇ Q ID NO: 3) or a significant portion (e.g. 100-500 nucleotides) thereof.
  • ASOs can be administered either in a single-stranded or double-stranded form, although the single-stranded form is preferable.
  • ASOs can be administered to an animal or a cell either in the form of a pharmaceutical composition comprising the ASO, as described herein.
  • the isolated polynucleotide of the invention can also be used as a template for expression of human Fezl protein, either in vitro or in vivo.
  • an isolated polynucleotide which does not comprise the intronic regions of FEZL such as an isolated polynucleotide which comprises a portion which is complementary to at least one strand of a cDNA generated using a spliced human mRNA encoding Fezl protein (e.g. a cDNA having the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 3).
  • the isolated polynucleotide of the invention When the isolated polynucleotide of the invention is used as a template for expression of human Fezl protein in vivo, the isolated polynucleotide has a sequence substantially homologous with at least nucleotide residues 112-456, nucleotide residues 1707-2510. and nucleotide residues 4912-5550 of at least one strand of SEQ ID NO: 1. If the cell in which Fezl protein is expressed is a mammalian cell, and especially if it is a human cell, it is not necessary to delete the intronic regions of FEZl from the isolated polynucleotide. Preferably, however, the mtromc regions of EEZ7 are deleted from the isolated polynucleotide prior to providing it to the cell.
  • the isolated polynucleotide of the invention is preferably provided to a cell in the form of an expression vector, wherein the region(s) encoding Fezl protein are operably linked with a promoter region.
  • the promoter region can be the human EEZ7 promoter region, or it can be substantially any other promoter region.
  • the promoter region of the expression vector is a constitutive promoter, an inducible promoter, or a tissue-specific promoter. Numerous constitutive promoters are known in the art and included within the scope of the invention.
  • Exemplary constitutive promoters include, for example, a retroviral LTR promoter, the cytomegalovirus immediate early promoter, the SN40 early promoter, the herpes simplex virus thymidine kinase promoter, an adenovirus-based promoter, elongation factor 1 alpha promoter, SV40-HTLV-1 LTR fusion promoter, and the CMV-beta actin enhancer fusion promoter.
  • Operable linkage of an isolated polynucleotide of the invention with an inducible promoter permits controlled expression of Fezl protein following delivery of the expression vector to a cell.
  • Such controlled expression is modulated by providing an inducer of the promoter to, or withholding or removing such an inducer from, the cell.
  • An example of an inducible promoter which can be operably linked to an isolated polynucleotide of the invention is a tetracycline promoter, which is well known in the art to be an inducible promoter.
  • tissue-specific promoter permits localization of expression of Fezl protein to a tissue of interest, thereby minimizing any side effects which can be associated with non- tissue- specific expression of Fezl protein.
  • the tissue- specific promoter may, for example, be selected from the group consisting of an epithelium-specific promoter, a tumor-specific promoter, a breast-specific promoter, a prostate-specific promoter, and an esophagus- specific promoter.
  • the prostate-specific antigen promoter can be operably linked to an isolated polynucleotide of the invention in order to achieve prostate-specific expression of Fezl protein.
  • the isolated polynucleotide of the invention can be provided to a cell, either in vitro or in vivo, using a wide variety of gene delivery vectors.
  • the identity of the vector is not critical; substantially any vector known in the art for delivering a nucleic acid to the interior of a cell can be used for this purpose.
  • Exemplary vectors include, but are not limited to naked D ⁇ A vectors, plasmids, condensed nucleic acids, projected nucleic acid-coated micro- or nano-particles. and virus vectors.
  • the invention also includes an animal cell which comprises an exogenous D ⁇ A molecule having at least one portion which is substantially homologous with at least the coding regions of the human EEZ7 gene.
  • the exogenous DNA molecule can comprise one, two, three, or more regions which, individually or together are substantially homologous with nucleotide residues 112- 456, nucleotide residues 1707-2510, and nucleotide residues 4912-5550 of at least one strand of S ⁇ Q ID NO: 1.
  • the exogenous DNA molecule comprises one region that is substantially homologous with at least one strand of S ⁇ Q ID NO: 2. More preferably, the exogenous DNA molecule is completely homologous with the coding regions of the human EEZ7 gene.
  • the exogenous DNA molecule comprises a promoter operably linked with the EEZ7 coding region(s), whereby Fezl protein is expressed in cells comprising the exogenous DNA molecule.
  • the cell can be a human cell, a non-human animal cell, or a non-animal cell, such as a plant cell, a yeast cell, a fungus cell, or a bacterium.
  • the cell can likewise be a cultured cell, a cell within the body of an animal, or a cell which is removed from the body of an animal for the purpose of providing the exogenous DNA molecule prior to returning the cell to the body of the same or a different animal.
  • the invention further relates to an animal comprising a cell which comprises an exogenous DNA molecule having at least one portion which is substantially homologous with at least the coding regions of the human EEZ7 gene.
  • the animal is a human which comprises a tissue which lacks a copy of the human wild type EEZ7 gene, such as certain tumor tissues.
  • Such animals e.g. mice
  • exon 1 of FEZl can be replaced with a neomycin- resistance cassette.
  • Embryonic stem cells of the animal are transfected using the targeting construct DNA vector, and cells are selected for neomycin resistance.
  • EEZ7 homologous recombination between the targeting construct DNA and one of the animal's genomic copy of the EEZ7 gene occurs. In rare instances, recombination of both EEZ7 copies can occur, but it is anticipated that most, if not all selected cells will be heterozygous for recombined EEZ7 -neomycin resistance gene, and will develop as heterozygous adult animals. These heterozygous animals exhibit characteristics attributable to animals having only a single functional EEZ7 gene per cell, such as abnormal cell or tissue differentiation, abnormal cell proliferation, increased incidence of cancer and other cell proliferative disorders, and uncontrolled gene expression. Furthermore, mating of heterozygous EEZ7 animals yields animals homozygous for the recombined EEZ7 -neomycin resistance gene (i.e. EEZ7
  • EEZ7 knockout animals exhibit traits characteristics attributable to the lack of a functional EEZ7 gene in the cells of the animal. Such characteristics include, for example, abnormal cell or tissue differentiation, abnormal cell proliferation, increased incidence of cancer and other cell proliferative disorders, and uncontrolled gene expression.
  • the invention also relates to an isolated Fezl protein.
  • the putative amino acid sequence of human Fezl protein (S ⁇ Q ID NO: 4) is shown in Figure 5D.
  • the isolated human Fezl protein is substantially purified.
  • the isolated human Fezl protein can be in the form of a suspension of the native or denatured protein in a liquid such as water, a buffer, or the like, a lyophilized powder, an immunogenic composition comprising the protein and one or more adjuvants or immunogenicity enhancers such as are known in the art. or a pharmaceutical composition as described elsewhere herein.
  • the isolated Fezl protein of the invention can be made by a variety of techniques.
  • the protein can be expressed in an in vitro expression mixture using an isolated polynucleotide of the invention.
  • the isolated polynucleotide of the invention can also be operably linked with a constitutive or other promoter, and the Fezl protein overexpressed in a human or non-human cell, and subsequently purified therefrom.
  • the Fezl protein can be purified using, for example, standard chromatographic techniques from a naturally occurring source of human Fezl protein (e.g. normal human brain or testes tissue).
  • the invention also includes fragments of the isolated Fezl protein of the invention.
  • fragments can be generated, for example, by expressing an isolated polynucleotide of the invention, wherein the polynucleotide encodes only a portion of human Fezl protein, or by proteolytic degradation of human Fezl protein.
  • the amino acid sequence of the isolated Fezl protein can comprise one or more conservative amino acid substitutions relative to SEQ ID NO: 4).
  • amino acids of the human Fezl protein can be substituted for other amino acids without appreciably affecting the biological activity of the protein.
  • amino acid sequence of the isolated Fezl protein of the invention is substantially homologous with SEQ ID NO: 4.
  • the hydropathic index of naturally occuning Fezl amino acid residues can be compared with those of potential substitute amino acid residues.
  • the significance of amino acid hydropathic index similarity between naturally occuning and potential substitute amino acid residues, as it relates to retention of biologic function of a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors. DNA. antibodies, antigens, and the like.
  • hydropathic index On the basis of their hydrophobicity and charge characteristics, as described (Kyte et al., 1982, J. Mol. Biol. 157:105). These hydropathic index values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8): tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • Amino acid residues can be substituted in place of other amino acid residues which having a similar hydropathic index without significantly affecting biological activity of the protein.
  • the substitute amino acid residue has a hydropathic index which differs from the hydropathic index of the naturally occurring amino acid residue by less than 2.0, preferably by less than 1.0, and more preferably by less than 0.5.
  • a substitute amino acid residue should have a hydropathic index in the range from 3.8 to -0.2, preferably in the range from 2.8 to 0.8, and more preferably in the range from 2.3 to 1.3.
  • arginine (+3.0) lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (0.0); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • Amino acid residues can be substituted in place of other amino acid residues having a similar hydrophilicity value without significantly affecting biological activity of the protein.
  • the substitute amino acid residue has a hydrophilicity value which differs from the hydrophilicity value of the naturally occurring amino acid residue by less than 2.0, preferably by less than 1.0, and more preferably by less than 0.5.
  • a substitute amino acid residue should have a hydrophilicity value in the range from 3.8 to -0.2, preferably in the range from 2.8 to 0.8, and more preferably in the range from 2.3 to 1.3.
  • amino acid substitutions can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity. hydrophilicity, charge, size, and the like.
  • conservative amino acid substitutions can include substitutions within the following groups: glycine. alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.
  • Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxvlation.
  • glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
  • enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes.
  • sequences which have phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
  • Analogs of such polypeptides include those containing residues other than naturally occuning L-amino acids, e.g. , D-amino acids or non-naturally occurring synthetic amino acids.
  • the isolated Fezl protein of the invention, and fragments thereof, are not limited to products of any of the specific exemplary processes listed herein.
  • the isolated Fezl proteins, and fragments thereof can incorporate amino acid residues which are modified without affecting activity.
  • the termini can be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C- termini from "undesirable degradation", a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound (e.g. as an anti-proliferative agent) by sequential degradation of the compound at a terminal end thereof.
  • Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect in vivo activities of the Fezl proteins or fragments thereof.
  • suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus.
  • suitable N- terminal blocking groups include C C 5 branched or non-branched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group.
  • Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal residue.
  • Suitable C-terminal blocking groups include esters, ketones. and amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH 2 ), and mono- and di-alkylamino groups such as methylamino, ethylamino. dimethylamino, diethylamino, methylethylamino. and the like are examples of C-terminal blocking groups.
  • Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the Fezl proteins, or fragments thereof, to yield desamino and descarboxylated forms thereof without affect on biological activity.
  • Fezl proteins, or fragments thereof can include one or more D-amino acid residues, or can comprise amino acids which are all in the D-form.
  • Retro-inverso forms of proteins peptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.
  • Acid addition salts of the Fezl proteins, or fragments thereof, of the present invention are also contemplated as functional equivalents.
  • a protein or peptide in accordance with the present invention can be treated with an inorganic acid such as hydrochloric, hydrobromic. sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic. maleic, fumaric, tartaric. citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic. p-toluenesulfonic, or salicylic acid to provide a water soluble salt of the peptide which is suitable for use as an anti-proliferative agent.
  • an inorganic acid such as hydrochloric, hydrobromic. sulfuric, nitric, phosphoric, and the like
  • an organic acid such as an
  • the isolated Fezl protein of the invention can be used to generate polyclonal or monoclonal antibodies using known methods.
  • administration of the Fezl protein of the invention to an animal can induce a soluble immune response against the protein or fragment in the animal.
  • the protein or fragment is mixed with an adjuvant or other immune system enhancer.
  • adjuvants include, but are not limited to. mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic poiyois. and polyanions, other peptides. and oil emulsions.
  • Antibodies which bind specifically with the Fezl protein or fragment can be identified and isolated using well known methods (see. e.g. Harlow et al.. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor, New York).
  • immortal hybridomas can be generated using known methods to provide a supply of such antibodies.
  • EEZ7 gene As described herein, reduced or no expression of the human EEZ7 gene has been demonstrated in numerous cancer cell lines and tumor samples. These data indicate that assessment of the level of EEZ7 gene expression in a cell or tissue of a human can indicate the cancerous state of the cell or tissue. Diagnostic techniques based on this relationship have the advantage that tumorigenesis can be detected in cells and tissues at an early stage, before other physiological changes associated with cancers can be detected in the same cells or tissues. Furthermore, these diagnostic techniques can be used to confirm or refute a preliminary diagnosis of tumorigenesis made by visual or cytological examination of potentially cancerous tissue.
  • the diagnostic methods described in this section are useful for diagnosing cancer in a body tissue of a human, particularly where the body tissue is an epithelial tissue.
  • the body tissue can be selected from the group consisting of a gastrointestinal tissue, esophageal tissue, gastric tissue, colon tissue, prostate tissue, breast tissue, a hematopoietic tissue, lung tissue, melanoma tissue, cervical tissue, and ovarian tissue.
  • the invention includes a method of determining the cancerous status of a sample tissue. This method comprises comparing EEZ7 expression in the sample tissue with EEZ7 expression in a control tissue of the same type. Decreased EEZ7 expression in the sample tissue, relative to EEZ7 expression in the control tissue, is an indication that the sample tissue is cancerous.
  • the sample tissue can be a phenotypically abnormal tissue (e.g. a biopsy sample obtained from a potentially cancerous lesion in a human tissue such as breast or prostate), or it can be a phenotypically normal tissue.
  • the control tissue is a non-cancerous tissue of the same type, and can be obtained from the same human from whom the sample tissue was obtained, or from one or more humans different than the one from whom the sample tissue was obtained. If a body of data exist or are created, from which a representative value for expression of FEZl in non-cancerous tissue of the same type as the sample tissue, then EEZ7 expression in the sample tissue can be compared with this representative value, rather than performing a separate determination of FEZl expression in the same or a different human.
  • EEZ7 in the sample tissue is compared with EEZ7 expression in a control tissue (or data set) by comparing the relative amounts of at least one indicator in the sample tissue and in the control tissue (or data set).
  • the indicator which is used can be any indicator which can be conelated with transcription of the EEZ7 gene in the tissue or with translation of this transcript is such tissue.
  • the indicator can be selected from the group consisting of a EEZ7 mRNA, a cDNA prepared using a EEZ7 mRNA, a DNA prepared by amplification of either of these, and Fezl protein.
  • the invention also includes another method of determining the cancerous status of a sample tissue.
  • This method comprises comparing the nucleotide sequence of a EEZ7 -associated polynucleotide obtained from the sample tissue with the nucleotide sequence of a control EEZ7 -associated polynucleotide. A difference between the nucleotide sequence of the FEZ -associated polynucleotide obtained from the sample tissue and the nucleotide sequence of the control EEZ -associated polynucleotide is an indication that the sample tissue is cancerous.
  • the FEZ1- associated polynucleotide may.
  • a chromosome for example, be one selected from the group consisting of at least a portion of a chromosome, a non-spliced mRNA. a partially spliced mRNA, a fully spliced mRNA, a cDNA prepared using a non-spliced mRNA, a cDNA prepared using a partially spliced mRNA, a cDNA prepared using a fully spliced mRNA. and a DNA prepared by amplification of any of these.
  • the FEZ1- associated polynucleotide can be DNA prepared by amplification of a cDNA prepared using a fully spliced mRNA obtained from a human, in which case, the control EEZ7- associated polynucleotide should be a DNA having the sequence S ⁇ Q ID NO: 3.
  • the EEZ7 -associated polynucleotide can be a DNA prepared by amplification of at least a portion of chromosome 8 of a human, in which case, the control EEZ -associated polynucleotide should be a DNA having the sequence S ⁇ Q ID NO: 1.
  • the sample and control tissues can both be obtained from the same human, in which case, the sample tissue should be a phenotypically abnormal portion of a body tissue of a human or a portion of the tissue in which tumorigenesis is anticipated, and the control EEZ7 -associated polynucleotide should be obtained from a phenotypically normal portion of the same body tissue or from a portion of the tissue in which tumorigenesis is not anticipated.
  • the sample and control tissues can also be obtained from the same tissue, but from different humans, in which case the control tissue should be obtained from a human whose relevant tissue is not cancerous.
  • the 'control tissue' can be a body of data collected from the relevant type of tissue obtained from a plurality of humans in whom the relevant tissue was not cancerous.
  • the nucleotide sequence of the sample EEZ7 -associated polynucleotide need be determined experimentally, and this sequence can be compared with a consensus or other sequence indicated by the body of data.
  • the EEZ7 gene sequence described herein S ⁇ Q ID NO: 1
  • the EEZ7 cDNA sequence described S ⁇ Q ID NO: 2
  • EEZ7 ORF sequence described herein can be used as the control EEZ7-associated polynucleotide sequence.
  • the invention includes yet another method of determining the cancerous status of a sample tissue.
  • certain mutations in the human EEZ7 gene lead to production of transcripts from this gene which have lengths which are different from the length of the wild type EEZ7 gene transcript.
  • This method conelates this transcript length difference with a cancerous state in a sample tissue.
  • This method comprises comparing the length of an EEZ7 -transcript-associated polynucleotide obtained from the sample tissue with the length of a control EEZ -transcript-associated polynucleotide. If the length of the EEZ7 -transcript-associated polynucleotide obtained from the sample tissue is less than the length of the control EEZ -transcript-associated polynucleotide.
  • the EEZ7 -transcript-associated polynucleotide may, for example, be selected from the group consisting of a fully spliced mRNA, a cDNA prepared using a fully spliced mRNA. and a DNA prepared by amplification of either of these.
  • the EEZ -transcript-associated polynucleotide is DNA prepared by amplification of a cDNA prepared using a fully spliced mRNA obtained from a human
  • the control EEZ -transcript-associated polynucleotide is DNA having the sequence S ⁇ Q ID NO: 2.
  • the EEZ7 -transcript-associated polynucleotide is fully spliced mRNA obtained from a human patient, and the control EEZ7 -transcript-associated polynucleotide is at least a portion of a nucleic acid which is complementary to S ⁇ Q ID NO: 2, whereby binding of the patient's mRNA and the control polynucleotide can be detected using standard RNA blot or Northern blot analytical techniques.
  • sample and control FEZ1- transcript-associated polynucleotides can be obtained from the same or different humans, and the control EEZ -transcript-associated polynucleotide can instead be a consensus or other relevant sequence described herein or formulated using FEZ1- transcript-associated polynucleotide sequences obtained from humans in whom the relevant tissue was not cancerous.
  • the invention includes still another method of determining the cancerous status of a sample tissue.
  • This method comprises assessing EEZ7 expression in the sample tissue.
  • a substantial absence of FEZl expression in the sample tissue is an indication that the sample tissue is cancerous.
  • EEZ7 expression can be assessed by assessing the presence or substantial absence of at least one indicator selected from the group consisting of a FEZl mRNA. a cDNA prepared using a EEZ7 mRNA. a DNA prepared by amplification of either of these, and Fezl protein.
  • the invention also includes another method of determining the cancerous status of a sample tissue.
  • This method comprises detecting abnormal splicing of a FEZl transcript in the sample tissue.
  • Abnormal splicing of the EEZ7 transcript is an indication that the sample tissue is cancerous.
  • Abnormal splicing of a EEZ7 transcript may, for example, be detected by assessing the ability of at least one exon boundary polynucleotide probe to anneal with a EEZ7 -transcript-associated polynucleotide with high stringency.
  • Such an exon boundary polynucleotide probe is capable of annealing with high stringency with terminal portions of two sequential EEZ7 exons when the terminal portions are adjacent, but not when the terminal portions are not adjacent.
  • such an exon boundary polynucleotide probe can comprise two portions, one portion which binds with high stringency with the 3 '-end of the first exon of a DNA, mRNA, or cDNA coding strand of FEZl, and another portion which binds with high stringency with the 5'-end of the second exon of a DNA, mRNA, or cDNA coding strand of FEZl. If the two portions of the probe are adjacent, then the probe will bind with high stringency with an mRNA, or with the coding strand of a cDNA generated using that mRNA. only if the two exons are adjacent in the mRNA or cDNA.
  • the probe will not bind with the mRNA, or the conesponding cDNA, with high stringency.
  • Design of such primers is well within the level of ordinary skill in the art, in light of the present disclosure.
  • the invention also includes an immunohistological method for detecting expression of Fezl protein in a cell or tissue sample obtained from a human patient.
  • This method involves use of an antibody preparation (e.g. a monoclonal or polyclonal antibody preparation) generated using the isolated Fezl protein of the invention (or a fragment thereof) according to standard antibody generating methods.
  • This preparation contains one or more types of antibodies which bind specifically with human Fezl protein.
  • the antibody preparation is contacted with the cell or tissue sample, and the Fezl -binding antibodies are labeled, either prior to or after contact with the sample. Non-specifically bound antibody is washed from the sample, and the presence of labeled antibody in or on the sample is assessed.
  • the presence of labeled antibody is an indication that the sample comprises human Fezl protein.
  • this immunohistological method can be used to detect Fezl expression, or a decrease of such expression, which is associated with an enhanced likelihood of tumorigenesis, for example.
  • Abnormal expression of the human EEZ7 gene is not merely a symptom of epithelial and other cancers in human tissues. It is also a contributing cause, and possibly the sole cause in some instances of tumorigenicity in those tissues. Inactivation of all genomic copies of the EEZ7 gene in one or more cells of a human tissue, especially an epithelial tissue, can lead to abnormal proliferation of those cells. Normal control of cell proliferation can be restored either by reactivating a genomic copy of the EEZ7 gene in abnormally proliferating cells or by providing at least one exogenous source of Fezl protein to abnormally proliferating cells.
  • the exogenous source of Fezl protein may, for example, be a nucleic acid encoding Fezl protein or a composition comprising Fezl protein.
  • the exogenous source of Fezl protein can be provided to the cells prior to tumorigenesis (i.e. for the purpose of inhibiting, delaying, or preventing tumorigenesis) or anytime after the onset of tumorigenesis (i.e. for the purpose of inhibiting, delaying, or preventing further abnormal proliferation of tumor cells or for the purpose of reversing abnormal proliferation).
  • the invention thus includes a method of modulating proliferation of a human cell having an altered EEZ7 gene.
  • This method comprises providing to the cell an exogenous source of Fezl protein.
  • Fezl protein When the protein is provided to the cell.
  • abnormal proliferation of the cell is inhibited, delayed, or prevented.
  • the cell to which the exogenous source of Fezl protein is provided can have one. two. or even more copies of an altered EEZ7 gene, and can have no normally-functioning copy of this gene. It is contemplated that, in most instances, this method will be employed in situations in which it is recognized that a tissue in a human patient comprises cells which do not express a wild type EEZ7 gene, or which express it at an abnormally low level. Expression of FEZl in a ceil is considered to be abnormally low when less than about 50. 49. 48, ..., 40. 39, 38, ..., 30, ..., 20 10. ...
  • the cell may, for example, be a cell which is recognizable as a tumor cell, a cell which is abnormally proliferating but not yet recognizable as a tumor cell, a metastatic cancer cell, a cell which is predisposed to abnormal proliferation but not yet recognizable as a tumor cell, or a cell which has an altered EEZ7 gene but is not proliferating abnormally at the time the exogenous source of Fezl protein is provided to the cell.
  • the cell is preferably an epithelial cell, such as a breast epithelial cell, a prostate epithelial cell, an esophageal epithelial cell, a lung epithelial cell, or an epidermal epithelial cell.
  • an epithelial cell such as a breast epithelial cell, a prostate epithelial cell, an esophageal epithelial cell, a lung epithelial cell, or an epidermal epithelial cell.
  • the altered EEZ7 gene may, for example, be one which is not transcribed in the cell, one which is transcribed to generate a transcript that is inconectly spliced, one which comprises at least one mutation which reduces or abolishes the normal function of Fezl protein, one which is transcribed but not translated, or one which has been partially or deleted from the genome of the cell.
  • the exogenous source of Fezl protein may, for example, be a composition comprising an isolated human Fezl protein of the invention, as described herein.
  • the Fezl protein can be a functional fragment or analog of Fezl protein (i.e. a fragment of Fezl or a peptidomimetic having structure similar to all or a portion of Fezl protein, wherein the fragment or analog exhibits one or more of the physiological activities of Fezl protein, such as inhibition of tubulin polymerization).
  • the Fezl protein is preferably a human Fezl protein or a human Fezl protein having one or more conservative amino acid residue substitutions.
  • the amino acid sequence of the Fezl protein is completely homologous with the amino acid sequence of the Fezl protein normally encoded by the EEZ7 gene of the cell.
  • the amino acid sequence of the Fezl protein is S ⁇ Q ID NO: 4.
  • the isolated Fezl protein provided to the cell may, as described herein, be expressed in vitro, isolated from an organism which has been transformed with a EEZ7 gene, or isolated from a naturaily-occuning source.
  • the Fezl protein can be isolated from cultured cells of a patient for provision to other cells of the same patient, either in vivo or ex vivo.
  • the Fezl protein can be isolated from cultured human or bacterial cells which have been transformed using an expression vector comprising a polynucleotide encoding at least the coding portion of a human EEZ7 gene (e.g. S ⁇ Q ID NO: 1, S ⁇ Q ID NO: 2. or S ⁇ Q ID NO: 3), and preferably at least the coding portion of a human EEZ7 gene obtained from the patient to whom the Fezl protein is to be administered.
  • the Fezl protein can be administered to a human in numerous pharmaceutical compositions.
  • the composition is one which is known in the art for providing proteins to the interior of a cell (e.g. liposomes. membrane vesicles, microspheres having an aqueous core, protein-coated projected particles, etc.).
  • the exogenous source of Fezl protein can also, for example, be an expression vector comprising a polynucleotide having at least one coding region which encodes a functional Fezl protein.
  • Fezl protein is provided to the cell.
  • the polynucleotide encodes a human Fezl protein or a human Fezl protein having one or more conservative amino acid residue substitutions.
  • the amino acid sequence of the Fezl protein is completely homologous with the amino acid sequence of the Fezl protein normally encoded by the EEZ7 gene of the cell.
  • the amino acid sequence of the Fezl protein is S ⁇ Q ID NO: 4.
  • the polynucleotide comprises a portion having the nucleotide sequence S ⁇ Q ID NO: 2. Also preferably, the polynucleotide comprises a portion which is substantially homologous, and more preferably completely homologous, with the wild-type genomic sequence of the EEZ7 gene of the patient to whose cell(s) the polynucleotide is provided.
  • the polynucleotide can comprise a portion which is substantially or completely homologous with S ⁇ Q ID NO: 1.
  • the polynucleotide may, of course, be an isolated polynucleotide of the invention, as described elsewhere herein, so long as the isolated polynucleotide encodes a functional Fezl protein.
  • Nucleic acid-containing vectors including expression vectors, are well known in the art, as are methods of targeting such vectors such that they provide the nucleic acid of the vector preferentially or exclusively to cells of certain types or to cells located primarily or only within certain tissues.
  • Exemplary expression vectors include both non-viral vectors (e.g. plasmids, naked DNA, DNA complexed with a polycation such as polylysine. and the like) and viral vectors such as retroviral. adenoviral. and adeno-associated viral vectors. The use of all such vectors is contemplated, and the selection of an appropriate vector is within the level of ordinary skill in the art.
  • the polynucleotide can be an expression vector in which the portion(s) of the polynucleotide which encode the Fezl protein is operably linked with a promoter.
  • the promoter can be a constitutive promoter, an inducible promoter, a tissue-specific promoter, or substantially any other promoter, although mammalian, and particularly human, promoters are prefened.
  • the promoter of the expression vector is a normal human EEZ7 promoter region.
  • the promoter is an inducible promoter, and this therapeutic method further comprises administering an inducer of the promoter to the cell to which the polynucleotide is provided.
  • the promoter is a tissue-specific promoter which normally promotes expression of genes operably linked therewith in an epithelial tissue.
  • an expressible portion of the EEZ7 gene is contained in the expression vector and is operably linked with a genetic element which can be used to cease EEZ7 expression. Numerous genetic elements of this type are known, including, for example, those associated with the Cre-loxP system (Pluck, Intl. J. ⁇ xp. Pathol.77:269-278; Li et al.. 1997, Human Gene Ther.
  • the invention also includes a method of preventing tumorigenesis in a human cell.
  • This method comprises providing to the cell an expression vector comprising a polynucleotide having at least one coding region which encodes a functional Fezl protein. Fezl protein is thereby expressed in the cell, and tumorigenesis is thereby prevented in the cell.
  • the cell may, for example, be one in which an altered EEZ7 gene has been detected, a cell of a tissue in which an altered EEZ7 gene has been detected, a normal cell in an individual predisposed to EEZ7 gene alteration (e.g. a human having a family history of FEZl gene alterations), or a normal cell in a normal individual.
  • the cell is an epithelial cell.
  • the polynucleotide can be any of those described herein for modulating proliferation of a human cell having an altered EEZ7 gene.
  • the invention also relates to a method of reversibly inducing proliferation of a cell. This method comprises providing an inhibitor of FEZl gene expression to the interior of the cell. Proliferation of the cell is induced when the inhibitor is present in the interior of the cell, but is not induced when the inhibitor is not present in the interior of the cell.
  • This method is useful to promote proliferation of desirable cells, either in vitro or in vivo. Examples of situations in which it would be advantageous to induce cell proliferation include, but are not limited to, when a tissue has been grafted from a location in one animal to another location in the same or a different animal (e.g.
  • a skin allograft or a bone marrow transplant when a mixture of desirable and undesirable cells has been treated to remove or kill undesirable cells (e.g. radiation therapy or chemotherapy of a partially cancerous tissue), or when healing of a wounded tissue is desired (e.g. healing of a skin puncture or incision).
  • undesirable cells e.g. radiation therapy or chemotherapy of a partially cancerous tissue
  • healing of a wounded tissue e.g. healing of a skin puncture or incision
  • the inhibitor used in this method can be an ASO, such as one of the isolated polynucleotides of the invention, or it can be a compound identified using one of the screening methods of the invention as an inhibitor of FEZl gene expression. If the inhibitor is capable of diffusing across the cell membrane, then it is not necessary to use a vector to deliver the inhibitor to the interior of the cell; otherwise, use of a vector to deliver the inhibitor to the interior of the cell. Any vector known in the art. such as any of those described herein, can be used for this purpose.
  • an ASO is prefened for reversibly inhibiting EEZ7 gene expression.
  • Useful ASO compositions are described elsewhere herein.
  • the ASO may, for example, be administered to the cell in the form a naked nucleic acid, a nucleic acid complexed with a polycationic or other condensing agent, a nucleic acid vector such as a plasmid or a virus vector, or the like.
  • the ASO can be provided to the interior of the cell directly, or an expression vector encoding the ASO can be provided to the interior of the cell. When such an expression vector is used, it is prefened that the expression of the ASO be regulatable.
  • the polynucleotide encoding the ASO can be operably linked with an inducible promoter, whereby the ASO is produced only when the inducer of the promoter is provided to the cell.
  • the expression vector can be incapable of being replicated.
  • replication-deficient vectors include, but are not limited to, plasmids which lack an origin of replication and replication-deficient virus vectors (e.g. replication- deficient adenovirus vectors).
  • the mechanism by which expression of the ASO is regulated is not critical; instead, it is important that expression of the ASO can be halted or severely limited when desired.
  • the polynucleotide or protein is preferably in the form of a pharmaceutical composition.
  • the invention includes a method of inhibiting tumorigenesis in a human. This method comprising administering to the human a compound selected from the group consisting of an inducer of FEZl gene expression, an enhancer of FEZl gene expression, a inhibitor of Fezl phosphorylation, an enhancer of phosphorylated- Fezl dephosphorylation, an agent that inhibits binding of Fezl with ⁇ Fl- ⁇ , and an agent that inhibits binding of Fezl with tubulin.
  • the invention encompasses the preparation and use of medicaments and pharmaceutical compositions comprising either Fez-1 protein, or another compound described herein as an active ingredient.
  • the isolated polynucleotide of the invention may, as described herein, be provided in the form of a nucleic acid vector, including, but not limited to, an expression vector.
  • the pharmaceutical compositions of the invention can consist of one or more active ingredients alone, in a form suitable for administration to a subject, or the pharmaceutical composition can comprise one or more active ingredients and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. Administration of one of these pharmaceutical compositions to a subject is useful for performing any of the methods of the invention, as described elsewhere in the present disclosure.
  • the active ingredient can be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to.
  • mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.
  • compositions that are useful in the methods of the invention can be prepared, packaged, or sold in formulations suitable for oral, rectal. vaginal, parenteral, topical, pulmonary, intranasal. buccal, ophthalmic, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • a pharmaceutical composition of the invention can further comprise one or more additional pharmaceutically active agents.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention can be made using conventional technology.
  • a formulation of a pharmaceutical composition of the invention suitable for oral administration can be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient.
  • Other formulations suitable for oral administration include, but are not limited to. a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
  • an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water.
  • a tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.
  • Compressed tablets can be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent.
  • Molded tablets can be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
  • Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to.
  • inert diluents include, but are not limited to, potato starch and sodium starch glycolate.
  • Known surface active agents include, but are not limited to, sodium lauryl sulfate.
  • Known diluents include, but are not limited to. calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate. calcium hydrogen phosphate, and sodium phosphate.
  • Known granulating and disintegrating agents include, but are not limited to. corn starch and alginic acid.
  • binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpynolidone, and hydroxypropyl methylcellulose.
  • Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
  • Tablets can be non-coated or they can be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and abso ⁇ tion of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate can be used to coat tablets.
  • tablets can be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4.265.874 to form osmotically- controlled release tablets.
  • Tablets can further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.
  • Hard capsules comprising the active ingredient can be made using a physiologically degradable composition, such as gelatin. Such hard capsules compnse the active ingredient, and can further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • a physiologically degradable composition such as gelatin.
  • Such hard capsules compnse the active ingredient, and can further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • Soft gelatin capsules comprising the active ingredient can be made using a physiologically degradable composition, such as gelatin.
  • a physiologically degradable composition such as gelatin.
  • Such soft capsules comprise the active ingredient, which can be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
  • Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration can be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
  • Liquid suspensions can be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions can further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions can further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpynolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxvpropylmethylcellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occuning phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol.
  • a partial ester derived from a fatty acid and a hexitol anhydride e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate. and polyoxyethylene sorbitan monooleate, respectively.
  • Known emulsifying agents include, but are not limited to, lecithin and acacia.
  • Known preservatives include, but are not limited to. methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol. sorbitol, sucrose, and saccharin.
  • Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
  • Liquid solutions of the active ingredient in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent.
  • Liquid solutions of the pharmaceutical composition of the invention can comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Powdered and granular formulations of a pharmaceutical preparation of the invention can be prepared using known methods. Such formulations can be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations can further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, can also be included in these formulations.
  • a pharmaceutical composition of the invention can also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
  • the oily phase can be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • compositions can further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occuning phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • emulsions can also contain additional ingredients including, for example, sweetening or flavoring agents.
  • a pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for rectal administration.
  • a composition can be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic inigation.
  • Suppository formulations can be made by combining the active ingredient with a non-initating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20°C) and which is liquid at the rectal temperature of the subject (i.e. about 37°C in a healthy human).
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides.
  • Suppository formulations can further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • Retention enema preparations or solutions for rectal or colonic irrigation can be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • enema preparations can be administered using, and can be packaged within, a delivery device adapted to the rectal anatomy of the subject.
  • Enema preparations can further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • a pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for vaginal administration.
  • a composition can be in the form of. for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a solution for vaginal irrigation.
  • Douche preparations or solutions for vaginal irrigation can be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • douche preparations can be administered using, and can be packaged within, a delivery device adapted to the vaginal anatomy of the subject.
  • Douche preparations can further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intravenous, intraanerial, intramuscular, or intrastemal injection and intravenous, intraarterial, or kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable canier, such as sterile water or sterile isotonic saline.
  • a pharmaceutically acceptable canier such as sterile water or sterile isotonic saline.
  • Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable formulations can be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations can further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • the pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution can be formulated according to the known art, and can comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations can be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3 -butane diol. for example.
  • Other acceptable diluents and solvents include, but are not limited to. Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation can comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
  • Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration can further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention can be prepared. packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • Such a formulation can comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low- boiling propellant in a sealed container.
  • a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low- boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers.
  • Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure.
  • the propellant can constitute 50 to 99.9% (w/w) of the composition, and the active ingredient can constitute 0.1 to 20% (w/w) of the composition.
  • the propellant can further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).
  • compositions of the invention formulated for pulmonary delivery can also provide the active ingredient in the form of droplets of a solution or suspension.
  • Such formulations can be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and can conveniently be administered using any nebulization or atomization device.
  • Such formulations can further comprise one or more additional ingredients including, but not limited to. a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate.
  • the droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.
  • the formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • Formulations suitable for nasal administration may. for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and can further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may. for example. 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and. optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration can comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient.
  • Such powdered, aerosolized, or aerosolized formulations, when dispersed preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and can further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention can be prepared. packaged, or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may. for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid canier.
  • Such drops can further comprise buffering agents, salts, or one or more other of the additional ingredients described herein.
  • Other ophthalmalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers: salts: thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • a pharmaceutical composition of the invention can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a "unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as. for example, one-half or one-third of such a dosage.
  • compositions which comprise an expression vector should be administered in an amount sufficient to provide at least one expression vector to the cell(s) being treated.
  • compositions comprising an expression vector are preferably administered in an amount sufficient to provide a two-, five-, ten-, or fifty-fold excess, or more, of the minimum recommended amount of the vector to individual cells.
  • Pharmaceutical compositions comprising an ASO should be administered in an amount sufficient to provide at least a quantity of ASO molecules equal to at least the expected or determined number of genomic copies of the ASO target or transcripts thereof.
  • compositions comprising an ASO are preferably administered in an amount sufficient to provide a two-, ten-, one hundred-, or one thousand-fold excess, or more, of the minimum recommended amount of the ASO to the target cells. It is understood that the ordinarily skilled physician or veterinarian will readily determine and prescribe an effective amount of the active ingredient(s) for performing the methods of the invention in a subject. In so proceeding, the physician or vetennarian may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. It is further understood.
  • kits comprising a pharmaceutical composition of the invention and an instructional material.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains a pharmaceutical composition of the invention or be shipped together with a container which contains the pharmaceutical composition.
  • the instructional material can be shipped separately from the container with the intention that the instructional material and the pharmaceutical composition be used cooperatively by the recipient.
  • the invention also includes a kit comprising a pharmaceutical composition of the invention and a delivery device for delivering the composition to a subject.
  • the delivery device can be a squeezable spray bottle, a metered-dose spray bottle, an aerosol spray device, an atomizer, a dry powder delivery device, a self-propelling solvent/powder-dispensing device, a syringe, a needle, a tampon, or a dosage measuring container.
  • the kit can further comprise an instructional material as described herein.
  • Identification of the human EEZ7 gene as a tumor suppressor gene provides a means for identifying compounds which induce cell proliferation. Also, because some altered EEZ7 genes associated with cancers in humans can prove to be capable of expression at normal, or near normal, levels in the presence of certain compounds, a method is provided for identifying such compounds, which can inhibit abnormal cell proliferation in cells having an altered EEZ7 gene, such as tumor cells and tissues.
  • the invention therefore includes a method of determining whether a test compound is an inducer of cell proliferation.
  • This metliod comprises incubating a cell which comprises a functional EEZ7 gene in the presence of the test compound and assessing expression of FEZl in the cell. If expression of FEZl in the cell is decreased relative to expression of FEZl in a cell of the same type incubated in the absence of the test compound, then the test compound is an inducer of cell proliferation.
  • Pa ⁇ icularly contemplated test compounds include isolated polynucleotides of the invention, as described herein. This method is therefore a useful way to identify ASOs which inhibit expression of FEZl and which therefore induce cell proliferation.
  • the cell which is used in this method can be substantially any cell which expresses a EEZ7 gene, such as one which transcribes the EEZ7 gene or one which both transcribes and translates FEZl.
  • the cell is a human cell, and it is more preferably an epithelial cell.
  • the cell used in this screening method be a cell of that certain type.
  • Expression of FEZl in the cell can be assessed by any known method of assessing gene expression. For example, the accumulated or steady-state amount of a transcript of FEZl or the rate of production of such a transcript in the cell of the screening method can be assessed using known methods. Alternately, the accumulated or steady-state amount of Fezl protein or the rate of production of Fezl protein can be assessed, likewise using known methods, including immunological methods involving an antibody of the invention.
  • the test compound can be administered to the cell in substantially any way.
  • the cell is incubated in a medium comprising the test compound.
  • the test compound does not readily pass from the medium to the interior of the cell (e.g. the test compound is a protein or a large nucleic acid in a form which does not normally cross cell membranes)
  • a vector can be used to deliver the test compound to the interior of the cell.
  • the screening method is intended to identify compounds which can be administered to a cell in the most convenient and physiologically acceptable form possible, it is prefened that the test compound not require a vector in order to reach the interior of the cell.
  • the invention also relates to a method of determining whether a test compound is effective to retard proliferation of a cell having an altered EEZ7 gene.
  • This method comprises incubating the cell having an altered EEZ7 gene in the presence of the test compound and assessing expression of FEZl in the cell. If expression of EEZ7 in the cell is increased, relative to expression of FEZl in a cell of the same type (i.e. also having the same altered EEZ7 gene) incubated in the absence of the test compound, then the test compound is effective to retard proliferation of a cell.
  • This result furthermore indicates that the test compound is a useful cancer therapeutic compound for treating cancer in a tissue which comprises cells of the type used in this screening assay.
  • This screening method is performed in substantially the same manner as the screening method described in the preceding paragraphs, except that the cell used in the screening method has an altered EEZ7 gene.
  • Fezl protein is a nucleic acid-binding protein.
  • This information indicates that it is possible to identify at least one nucleic acid sequence with which Fezl protein binds by contacting Fezl protein with a test nucleic acid sequence and assessing whether the protein and the nucleic acid form a complex. Any known method for detecting such complexes can be used, including, but not limited to, nucleic acid footprint methods, altered gel electrophoresis mobility methods, altered chromatographic mobility methods, immunological methods involving an antibody of the invention.
  • a nucleic acid comprising that sequence can be used as an inducer of cell proliferation by delivering such a nucleic acid to a cell comprising a functional Fezl protein.
  • the nucleic acid binds with the Fezl protein in the cell, preventing Fezl from binding with its normal physiological binding partner, and thereby inducing cell proliferation.
  • the nucleic acid is preferably used in great excess (e.g. 10-. 100-, or 1000-fold or more excess) of the intracellular concentration of Fezl protein.
  • the screening methods of the invention can be used to identify anti- cancer therapeutic compounds for administration to a human afflicted with a cancer by identifying test compounds a inducers of altered EEZ7 gene expression. Because the human EEZ7 gene can be altered in numerous ways in various cancers and in different individuals, it is advantageous to perform the screening methods of the invention using cells obtained from the patient to be treated. In order to facilitate such treatment, components used in these assay methods can be conveniently packaged in the form of a kit comprising a plurality of candidate anti-cancer therapeutic compounds and a reagent for assessing expression of FEZl in the patient's cells.
  • the reagent is an isolated polynucleotide which anneals with high stringency with a human EEZ7 gene, such as an isolated polynucleotide which anneals with high stringency with at least twenty consecutive nucleotide residues of at least one strand of S ⁇ Q ID NO: 1.
  • the reagent is the antibody of the invention, as described herein.
  • the invention includes a screening method for determining whether a test compound is useful for alleviating a disorder associated with abenant tubulin polymerization. This method comprising comparing
  • tubulin polymerization in a first assay mixture which comprises tubulin, Fezl, and the test compound and (ii) tubulin polymerization in a second assay mixture which comprises tubulin and Fezl, but which does not comprise the test compound.
  • a difference e.g. a difference between the rate of tubulin polymerization in the first and second assay mixtures or a difference between the extent of tubulin polymerization in the first and second assay mixtures between tubulin polymerization in the first and second assay mixtures
  • the first and second assay mixtures are substantially identical, but for the presence or absence of the test compound.
  • disorders which can test compounds can be tested include both tubulin hyperpolymerization disorders and tubulin hypopolymerization disorders.
  • the disorder can be one selected from the group consisting of a disorder associated with abenant initiation of mitosis, a disorder associated with abenant modulation of the rate and stage of mitosis, a disorder associated with abenant modulation of the initiation and rate of cell proliferation, a disorder associated with abenant modulation of the initiation and rate of cell growth, a disorder associated with abenant modulation of cell shape, a disorder associated with abenant modulation of cell rigidity, a disorder associated with abenant modulation of cell motility, a disorder associated with abenant modulation of the rate of cellular DNA replication, a disorder associated with abenant modulation of the stage of cellular DNA replication, a disorder associated with abenant modulation of the intracellular distribution of organelles, a disorder associated with abenant modulating the metastatic potential of a cell, and a disorder associated with abenant modulation of the
  • test compound used in this screening method can be substantially any compound.
  • Compounds which are anticipated to be particularly likely to be useful for alleviating such disorders include ones selected from the group consisting of a fragment of Fezl, a peptidomimetic of a fragment of Fezl, a fragment of tubulin, a peptidomimetic of a fragment of tubulin, a fragment of EFl- ⁇ , and a peptidomimetic of a fragment of EF 1 - ⁇ .
  • the invention includes another screening method for determining whether a test compound is useful for alleviating a disorder associated with abenant phosphorylation of Fezl .
  • This method comprises comparing
  • phosphorylation of Fezl in a second assay mixture which comprises Fezl. the kinase, and the phosphate source, but which does not comprise the test compound.
  • a difference between phosphorylation of Fezl in the first and second assay mixtures e.g. a difference in the rate or degree of phosphorylation in the first and second assay mixtures
  • This screening method can be used to assess the utility of compounds for alleviating the same disorders refened to above.
  • the invention includes yet another screening method for determining whether a test compound is useful for alleviating a disorder associated with abenant phosphorylation of Fezl . This method comprising comparing
  • a difference between phosphorylation of Fezl in the first and second assay mixtures is an indication that the test compound is useful for alleviating the disorder.
  • This screening method can be used to assess the utility of compounds for alleviating the same disorders refened to above.
  • the invention also includes a method of determining whether a test compound is useful for alleviating a disorder associated with abenant binding of Fezl with a protein with which Fezl normally binds, the method comprising comparing
  • a difference between (e.g. the rate or degree of) binding of Fezl and the protein in the first and second assay mixtures is an indication that the test compound is useful for alleviating the disorder.
  • the protein can, for example, be selected from the group consisting of tubulin and EFl- ⁇ . This screening method is useful for assessing the utility of a test compound for alleviating a disorders such as one of tumorigenesis, tumor survival, tumor growth, and tumor metastasis.
  • the invention includes a method of determining whether a test compound is an inhibitor of cell proliferation. This method comprises incubating a cell which comprises a functional FEZ7 gene in the presence of the test compound and assessing expression of FEZl in the cell. If expression of FEZl in the cell is increased, relative to expression of FEZl in a cell of the same type incubated in the absence of the test compound, then this is an indication that the test compound is an inhibitor of cell proliferation.
  • the human EEZ7 gene has been discovered to be a rumor suppressor gene.
  • inactivation of this gene, or inhibition of expression of this gene leads to the enhanced rate of cell proliferation associated with cancer.
  • enhanced cell proliferation is desirable.
  • some in vitro cell culture methods are limited by the rate of cell proliferation and by effects of cell density on this rate.
  • certain medical procedures such as in bone marrow transplants and skin allografts. it is desirable that cells proliferate at a greater-than-normal rate for a period and subsequently proliferate only at a normal rate.
  • Providing a cell with an inhibitor of FEZl expression enhances the rate of proliferation of the cell, and this technique can be used to improve a vanety of known methods in which the rate of cell proliferation was a limiting factor.
  • an inhibitor of FEZl expression in a cell culture medium, or by treating cells (e.g. human epithelial cells) growing on or in such medium with such an inhibitor, the in vitro rate of cell proliferation can be increased, permitting faster and denser cell growth than would otherwise be possible.
  • the rate of proliferation of those cells can be increased.
  • This method can be used, for example to enhance graft integration into the graft site or to improve reestablishment of bone manow in an individual who has been subjected to levels of radiation or cytotoxic chemicals that are sufficient to cause bone manow loss.
  • Local administration of the inhibitor to the tissue(s) or region(s) in which enhanced cell proliferation is desired minimizes undesirable cell proliferation in other tissues and at other body regions. Discontinuing administration of the inhibitor leads eventually to normal cell proliferation of treated cells, owing to degradation of the inhibitor.
  • cells obtained from a human can be treated ex vivo with an inhibitor of FEZl expression to enhance their rate of proliferation prior to implanting those cells within the same human from which they were obtained or within a different human.
  • the same or a different inhibitor of FEZl expression can be administered, locally or systemically, to the human cell recipient in order to maintain the enhanced rate of proliferation of the treated cells, or the cells can instead be permitted to retain their enhanced rate of proliferation only so long as the inhibitor delivered to them ex vivo endures. In either event, the ex vivo treated cells assume a normal rate of proliferation after the inhibitor(s) are degraded and not replaced.
  • the inhibitor of FEZl expression can optionally be a molecule which is capable of being replicated in a human cell, such as a virus vector encoding such an inhibitor, for example.
  • the inhibitor is preferably not capable of being replicated in a human cell.
  • the inhibitor be provided to the cells in the form of a vector which comprises a polynucleotide encoding the inhibitor, and that the polynucleotide be operably linked to an inducible promoter, so that production of the inhibitor can be initiated and concluded by administration and withholding, respectively, of the inducer of the promoter.
  • Kits of the Invention includes various kits which comprise any two or more of the isolated polynucleotides of the invention, the isolated Fezl proteins of the invention, pharmaceutical compositions, and instructional materials which describe use of these polynucleotides and proteins to perform the diagnostic, therapeutic, or screening methods of the invention.
  • exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure. Each of these kits is included within the invention.
  • kits of the invention are kits for amplifying at least a portion of a human EEZ7 gene.
  • This kit comprising a first isolated polynucleotide and a second isolated polynucleotide, wherein the first isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of one strand of a human EEZ7 gene and the second isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of the other strand of the gene.
  • the first isolated polynucleotide can be one which anneals with high stringency with at least twenty consecutive nucleotide residues of the coding strand of S ⁇ Q ID NO: 1
  • the second isolated polynucleotide can be one which anneals with high stringency with at least twenty consecutive nucleotide residues of the non-coding strand of S ⁇ Q ID NO: 1.
  • This kit can further comprise other components of a reaction mixture for amplifying a region of a nucleic acid, such as a DNA polymerase (e.g. Thermits aquaticus DNA polymerase) or deoxyribonucleotides.
  • this kit can include an instructional material which describes the polynucleotides as being useful for amplifying a portion of the gene or which describe how to perform such an amplification.
  • a second example of a kit of the invention is a kit for amplifying at least a portion of a cDNA generated from a transcript of a human EEZ7 gene.
  • This kit comprises a first isolated polynucleotide and a second isolated polynucleotide.
  • the first isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of the cDNA
  • the second isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of the cDNA.
  • the first isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of the coding strand of S ⁇ Q ID NO: 1
  • the second isolated polynucleotide anneals with high stringency with at least twenty consecutive nucleotide residues of the non-coding strand of S ⁇ Q ID NO: 1.
  • the EEZ7 Gene at Chromosome Location 8p22 Encodes a Leucine-Zipper Protein, and its Expression Is Altered in Multiple Human Tumors Loss of heterozygosity (LOH) at 8p22 is a common characteristic of epithelial tumors, including breast, prostate, and esophageal carcinomas.
  • LH heterozygosity
  • EEZ7 encodes a leucine-zipper protein having substantial amino acid sequence similarity to the DNA-binding protein designated Atf-5. EEZ7 expression could not be detected in more than 60% of epithelial tumors and tumor cell lines of various types. Furthermore, transcript analysis of EEZ7 -expressing tumor cells indicated the presence of mutations in FEZl. as evidenced by the presence of sequence abnormalities in the EEZ7 transcript, and the presence of frame-shift mutations, as evidenced by the presence of truncated EEZ7 transcripts. Based on the results described in this Example, it is concluded that alteration or inactivation of FEZl is involved in development of multiple human tumors, including epithelial tumors.
  • Esophageal cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum.
  • Prostate cancer cell lines, breast cancer cell lines, hematological cell lines, and HeLa cells were obtained from the American Type Culture Collection and were cultured as described (Negrini. 1996).
  • Tumor and non-tumor tissue samples were obtained from 72 patients afflicted with primary esophageal cancers, 39 patients afflicted with breast cancers, 24 patients afflicted with prostate cancers, and 8 patients afflicted with ovarian cancers.
  • Chromosomal DNA was isolated from 53 primary esophageal squamous cell tumors and from matched normal tissue samples obtained from the same patients. These DNA samples were analyzed for allele loss at 22 microsatellite loci on chromosome 8p.
  • PCR amplification of microsatellite loci using FAM- or TET-labeled primers were performed as described (Niederacher et al.. 1997, Genes Chromosom. Cancer 18:181), with minor modifications. Briefly, PCR was performed using AmpliTaqTM Gold (Perkin Elmer Cetus, Norwalk, CT), using the following reaction conditions. After heating the reaction mixture to 95°C for 12 minutes, a total of 30 PCR cycles were performed. The first 10 cycles consisted of maintaining the reaction mixture at 94°C for 15 seconds, at 55-58°C for 15 seconds (to anneal DNA strands), and at 72°C for 30 seconds.
  • the next 20 cycles consisted of maintaining the reaction mixture at 89°C for 15 seconds, at 55-58°C for 15 seconds (to anneal DNA strands), and at 72°C for 30 seconds. Following these 30 cycles, the reaction mixture was maintained at 72°C for 30 minutes. Following heat denaturation, the amplified reaction mixtures were loaded on a 6% (w/v)polyacrylamide denaturing gel on the Applied Biosystems model 373 DNA sequencer. Data collection and fragment analysis were performed using ABI PrismTM Genescan and ABI PrismTM Genotyper Analysis Software (Perkin Elmer Cetus, Norwalk. CT; Applied Biosystems. Inc., Foster City, CA).
  • LOH was detected as reduction by more than 50% of an allele peak signal in DNA obtained from a tumor sample, relative to the peak signal of the same allele in conesponding normal tissue. If a tumor sample demonstrated 40-60% reduction of an allele peak signal, relative to the conesponding normal tissue, the analyses were repeated two more times, and average reductions were used as final data.
  • Yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) contigs of the region of the genome near the D8S261 marker were constructed. The relative positions of the YAC and BAC contigs. relative to certain microsatellite loci, are indicated in Figure lC. Human chromosome 8p BAC DNA samples were sequenced using primers T7 and SP6 (Research Genetics, Huntsville, AL). Southern blot hybridization and PCR analysis indicated that BAC clones overlapped, and contigs were constructed.
  • PCR amplification was performed using STS (sequence tagged sequences) primers in order to screen a human YAC library obtained from Research Genetics (Huntsville. AL).
  • STS sequence tagged sequences
  • a mixture of YAC clones was embedded in an agarose gel and separated by pulse-field gel electrophoresis (PFGE), as described (Ausubel et al.. 1989. In: Cunent Protocols in Molecular Biology. Wiley-Interscience. New York; Bookstein. et al.. 1994, Genomics 24:317).
  • PFGE pulse-field gel electrophoresis
  • YAC DNA was transfened to a nylon membrane in the presence of 0.4 molar sodium hydroxide, and the membrane was hybridized using human genomic DNA.
  • DNA from individual YAC clones was digested within the gel using Mbol fox four hours at 37°C. Digested YAC clone DNA was extracted from the gel using a Gene Clean IIITM kit obtained from BIO 101. Inc. (La Jolla. CA), per the kit instructions.
  • cDNAs were synthesized by reverse transcription of prostate poly(A) ' RNA using Not7-primer adaptor/oligo-dT primers according to the GC rich protocol (SuperscriptTM Plasmid system: Gibco-BRL, Grand Island, ⁇ Y). A Sal I adaptor (Gibco-BRL, Grand Island. NY) was ligated to the cDNAs, and those cDNAs were subjected to twenty cycles of PCR amplification using adapter primers.
  • Blocking, hybridizing, and washing methods were adapted from described procedures (Bookstein et al., 1997, Br. J. Urol. 79(Suppl. 1):28; Bova et al., 1996, Genomics 35:46; MacGrogan et al., 1996. Genomics 35:55; Cher et al.. 1994, Genes Chromosom. Cancer 11 :153; Bookstein, et al.. 1994, Genomics 24:317; Akiyama et al., 1997, Cancer Res. 57:3548).
  • PCR-amplifiable cDNA 10 microliters of an avidin-coated magnetic bead suspension (DynabeadsTM M- 280; Dynal, Lake Success, NY) were mixed with 100 micrograms of sonicated salmon sperm DNA at room temperature for 30 minutes.
  • the beads were pre-washed with TE buffer containing 1 molar NaC 1 , and were then incubated with complete hybridization reaction mixtures in 200 microliters of the same buffer at room temperature for 30 minutes.
  • the beads were collected by using a magnetic concentrator (Dynal, Lake Success, NY), and the supernatant was removed.
  • cDNA was eluted from beads by mixing the beads with 100 microliters of 50 millimolar NaOH for 15 minutes and then neutralizing the mixture using 100 microliters of 1 molar Tris-HCl buffer at pH 7.5.
  • cDNA was purified using a PCR purification column (QiagenTM, Chatsworth. CA), per the manufacturer's instructions.
  • cDNA was re-amplified by PCR using the same methods and the same conditions. The resulting amplified cDNA products were purified and blocked, and a second round of cDNA selection was performed as described herein.
  • Amplified cDNA was digested using restriction endonucleases Sail and Not7, cloned directly into pSPORTl vector (Gibco-BRL, Grand Island, ⁇ Y), and used to transform E. coli cells. CpG island cloning and shotgun sequencing were performed using this cD ⁇ A-containing vector. Using these methods, 87 potentially expressed clones were mapped in the YAC contig, as indicated in Figure lC.
  • BAC D ⁇ As were digested using restriction endonucleases BssHII and S ⁇ c77. which specifically cleave CpG islands. After digestion with Sai ⁇ AI, the cleavage products were ligated into a pBK-CMV vector (Stratagene. La Jolla. CA), as described ( ⁇ lvin et al.. 1992. In: Techniques for the analysis of complex genomes: Transcribed sequences in YACs. Anand, ⁇ d.. Academic Press. London, p. 155).
  • Shotgun sequencing was performed as described (Inoue et al.. 1997, Proc. ⁇ atl. Acad. Sci. USA 94:14584). Six hundred clones per BAC were picked and sequenced to identify candidate cD ⁇ A sequences. cD ⁇ A selection were performed for three YAC templates, as indicated in Figure lC. Four hundred clones per YAC were picked up from the cD ⁇ A selected libraries, and all the clones were sequenced with vector primers. The sequences were analyzed using the BLAST computer software and the ⁇ CBI/BLAST database in order to exclude ribosomal or mitochondria-related genes. Fifty percent of clones were ribosomal or mitochondria-related genes, and the remainder were classified and were analyzed.
  • BACs Two candidates CpG islands were identified from the region near marker D8S233 by CpG island cloning.
  • the BACs were partially sequenced by the shotgun method to determine the presence of sequences matching expressed sequence tags (ESTs) in the nucleotide/EST database of NCBI/BLAST, and two ESTs from the BAC genomic region were thereby identified.
  • ESTs expressed sequence tags
  • cDNA was synthesized using 2 micrograms of total RNA obtained from human brain, esophagus, or tumor cells or from 150 nanograms of poly(A)" RNA obtained from one of these cell types using the Superscript IITM plasmid system (Gibco-BRL, Grand Island, NY).
  • the cDNA and an adaptor (Catalog #K1802-1 ; Clontech, Inc.. Palo Alto, CA) were ligated to generate RACE templates, and the templates were used in PCR amplification of the cDNA.
  • the chromosomal location of the F37 gene was confirmed by identification of the presence of the F37 gene sequence at 8p22 in a radiation hybrid panel designated Gene Bridge 4TM (Research Genetics, Huntsville. AL).
  • Full-length and 3'-truncated FEZl cDNAs were ligated to a expression vector pcDNA3HisA (Invitrogen, Carlsbad, CA) and cloned by RT-PCR, using human brain cDNA as a template.
  • the entire nucleotide sequence of the insert cDNA was verified by DNA sequencing.
  • the truncated cDNAs (nucleotides 1-1128 in the EEZ7 ORF) lacked the portion of the ORF located 3'- with respect to the leucine zipper region.
  • FIG. 2C An image of the SDS-PAGE results is shown in Figure 2C.
  • the protein in lane 1 of that SDS-PAGE was translated from a pcDNA vector which comprised a truncated EEZ7 cDNA lacking the portion of the cDNA located 3'- with respect to the leucine zipper region.
  • the protein in lane 2 was translated from a pcDNA vector which comprised full-length EEZ7 cDNA.
  • the protein in lane 3 was translated from a pcDNA vector having no insert.
  • the protein in lane 3 was translated from a pG ⁇ X vector which comprised a truncated EEZ7 cDNA lacking the portion of the cDNA located 3'- with respect to the leucine zipper region.
  • the nucleotide sequence of the EEZ7 gene open reading frame was analyzed in samples obtained from 194 cancer tissues, regardless of the whether or not EEZ7 was expressed in the tissue.
  • the sampled tissues were obtained from 72 primary esophageal cancers, 18 esophageal cancer cell lines, 24 primary prostate cancers, 3 prostate cancer cell lines, 39 primary breast cancers, 25 breast cancer cell lines. 8 primary ovarian cancers. 4 leukemic cell lines, and one cervical cancer cell line.
  • Nucleotide sequence information was obtained by PCR and sequencing. Eleven pairs of PCR primers, as described herein in the Primer Nucleotide Sequence Table, were used to amplify EEZ7 coding exons 1-3.
  • Genomic PCR was performed using the same conditions described herein for LOH studies, except that 4% DMSO (w/w) was added to the reaction mixture, and PCR amplifications were performed for 35 cycles, the additional 5 cycles being the same as the 20 cycles described herein.
  • DNA sequencing was performed directly using the purified PCR amplification products. Sequencing reactions and analyses were performed by using the ABI Prism BigDyeTM terminator reaction chemistry on the ABI PrismTM 377 DNA sequencing system (Applied Biosystems. Inc.. Foster City, CA). Sequence data were confirmed by sequencing of duplicate PCR amplification products and by sequencing anti-sense strands using reverse primers. The results of the experiments presented in this Example are now described.
  • tissue samples obtained from 23 of 53 patients exhibited loss of an allele at one or more loci on 8p, as indicated in Figures 1A and IB.
  • tissue samples obtained from patient E26 exhibited LOH at the markers designated D8S264.
  • LPL and D8S136 and allelic retention at the marker designated FGFR1.
  • Tissue samples obtained from patient E46 exhibited LOH at the markers designated D8S264 and D8S136, and the markers designated LPL and FGFR1 loci were homozygous, meaning that loss of an allele from one chromosome could not be detected if it occuned.
  • F37 cDNA comprises a 1791 base pair open reading frame (ORF) which encodes a 597 amino acid residue protein having a molecular weight of approximately 67 kilodaltons.
  • ORF open reading frame
  • Homology searching of protein sequence databases indicated the amino acid sequence of F37 comprises a leucine-zipper motif, and that this region has 32% identity (68% similarity) to the DNA-binding domain of a c AMP -responsive activating-transcription factor designated Atf-5 (Hai et al., 1989. Genes Develop. 3:2083).
  • the homology search also indicated that the F37 protein has 38% identity to the protein designated KIAA0552. which consists of 673 amino acids (Nagase et al, 1998. DNA Res. 5:31-39).
  • Motif analysis software (Searching Protein and Nucleic Acid Sequence Motifs in Genome Net) predicted a cAMP-dependent phosphorylation site, located at Ser 29 of F37. and a predicted tyrosine-kinase phosphorylation site, located at Tyr 67 of F37.
  • the ORF comprised three coding exons.
  • the F37 gene was designated EEZ7 (F37/ ⁇ sophageal cancer gene encoding leucine-zipper motif).
  • the putative amino acid sequence of Fezl, the protein encoded by EEZ7 is listed in Figure 2A. Nucleotide residues around the first methionine codon in EEZ7 cDNA were matched using the Kozak recognition rule (Kozak. 1989, J. Cell. Biol.
  • EEZ7 gene expression was almost ubiquitous in normal tissues. EEZ7 expression was most prominent in testes, as indicated in Figure 2D. EEZ7 gene expression was analyzed by Northern blotting and by RT-PCR amplifications in human tumor tissue samples, including 41 cancer-derived cell lines and 25 primary tumors, as indicated in Figure 3 A and summarized in Table 1. EEZ7 expression was undetectable in 31 cancer cell lines (76%) and 16 primary tumor samples (64%). EEZ7 expression was not detected in any of the 15 breast cancer cell lines studied or in any of the 10 primary breast tumor samples studied. However, EEZ7 was expressed in normal tissues.
  • EEZ7 Expression was detected by Northern blot or RT-PCR (indicated by *). 2 In El 6. E26 and E41. normal tissues from a single patients' organs were analyzed, and did not exhibit alterations in the coding region sequences, n addition, the coding sequences from normal prostate as well as the other four samples from normal esophagus were analyzed, and no alterations were found except that one of twelve sequenced clones from testis cDNA showed a deletion of nucleotide.
  • FEZl expression was assessed in normal breast epithelial cells and fibroblasts and in normal prostate epithelial cells (these three types of cells were obtained from Clonetics, San Diego, CA). RT-PCR amplification indicated that EEZ7 was expressed in these three types of normal cells. No EEZ7 expression could be detected in breast and prostate (LNCaP) cancer cells.
  • the nucleotide sequence of the EEZ7 gene ORF was analyzed in a total of 194 cancer tissue samples, regardless of whether EEZ7 was expressed in the tissue. These tissue samples included 72 primary esophageal cancer tissue samples. 18 esophageal cancer cell lines, 24 primary prostate cancer tissue samples, 3 prostate cancer cell lines. 39 primary breast cancer tissue samples, 25 breast cancer cell lines, 8 primary ovarian cancer tissue samples. 4 leukemic cell lines, and one cervical cancer cell line. Tliree point mutations were identified, two in two primary esophageal cancer tissue samples, and one in a prostate cancer cell line, as indicated in Figure 3B. These point mutations are summarized in Table 2. Table 2
  • a point mutation resulted in an amino acid substitution of serine (normal) to proline (mutant) at amino acid residue 29.
  • Amino acid residue 29 is, as described herein, a predicted cAMP-dependent kinase phosphorylation site.
  • a second point mutation resulted in a different amino acid substitution, namely lysine (normal) to glutamate (mutant) at amino acid residue 1 19.
  • the third point mutation which was detected was a change of a codon encoding a glutamine residue in the normal EEZ7 transcript to a stop codon at codon 501 in a prostate cancer cell line designated PC3.
  • This mutation resulted in a EEZ7 transcript which encoded a putative 166 amino acid residue protein lacking the normal carboxyl terminal region of wild type F ⁇ Z1 protein.
  • Northern blotting, RT-PCR, and nucleotide sequencing revealed that these three mutated DNA sequences were expressed in the patients and cells in which they were identified.
  • EEZ7 ORF sequences did not differ from wild type sequences in samples obtained from normal brain and prostate tissues (obtained from Clontech, Inc., Palo Alto, CA), from normal esophagus tissue samples obtained from seven individuals, or from matched normal cDNA obtained from patients ⁇ 16, E26, and E41.
  • One of twelve clones derived from testes cDNA samples obtained from ClonTech Inc.. Palo Alto.
  • CA cDNA exhibited a deletion at nucleotides 1441-1527 in the ORF.
  • the positions of the first and last nucleotides of deletions are shown according to the nucleotide number counted from first coding codon.
  • Zip(+) means a protein comprising a leucine-zipper region
  • Zip(-) means a protein not comprising a leucine-zipper region.
  • Nucleotide residues are numbered relative to the position of the first nucleotide residue of the first codon of FEZl (i.e. residue 1). Nucleotide sequences flanking cD As deletion endpoints of are indicated. Upper case letters indicate nucleotide residues which are present in truncated cDNAs. Lower case letters in parenthesis indicate nucleotide residues not present in truncated cDNAs. Underlined characters indicate conserved nucleotide residues at donor/acceptor sites. The deletion in a results in a frame-shift which encodes a protein having a putative molecular weight of 8.6 kilodaltons.
  • the allelic expression status of FEZl was analyzed using a polymo ⁇ hic site in the 3'-noncoding cDNA region, namely the 2134th nucleotide residue of FEZl cDNA, numbered from the first nucleotide residue of the first codon.
  • the EEZ7 gene was transcribed from both alleles, i.e. it was not imprinted.
  • Example 2 Effect of Fezl Expression on Growth of Cells of Breast Cancer Cell Line MCF7
  • MCF7 available from American Type Culture Collection, Gaithersburg, MD; accession number HTB-22
  • MCF7 available from American Type Culture Collection, Gaithersburg, MD; accession number HTB-22
  • EEZ7 expression inhibited cell growth in vitro and in vivo.
  • MCF7 cells were stably transfected using a pTet-OffTM plasmid vector (ClonTech, Palo Alto CA; GenBank Accession number U89929) in which at least the coding portion of the EEZ7 gene was operably linked with the tetracycline-responsive element and promoter of the vector.
  • Cells were maintained in DM ⁇ M medium supplemented with 2 micrograms per milliliter doxycycline (Sigma Chemical Co., St. Louis, MO. catalog number D-9891) and 10% (v/v) certified fetal bovine serum (FBS; ClonTech).
  • Stable transfectants were made by maintaining transfected cells for about 2 weeks in medium containing hygromycin (Gibco, Grand Island, NY) at a concentration of about 200 micrograms per milliliter, beginning 36 hours after transfection.
  • hygromycin Gibco, Grand Island, NY
  • Four well-isolated transfectant clones were selected and designated clones 15, 18, 54 and 118. These clones were cultured in tetracycline-free medium comprising 10% (v/v) serum medium for 72 hours in order to induce expression of FEZl.
  • tetracycline and doxycycline were used interchangeably, because the tetracycline-responsive elements are substantially equally responsive to tetracycline and doxycycline.
  • the effect of FEZl expression on in vitro cell growth of MCF7 cells was analyzed using the CellTiter 96TM AQueous non-radioactive cell proliferation assay obtained from Promega Co ⁇ oration (Madison. WI) per the supplier's instructions.
  • the absorbance of the MTS compound of the assay system at 490 nanometers exhibited a linear conelation between the number of MCF7 cells in a range between 10 2 and 10 4 cells, as confirmed by cell counting in which dead cells were excluded the dead cells by trypan blue staining.
  • Cells of clones 15, 18, 54, and 118 were seeded in wells of 96- well plates containing tetracycline-free medium supplemented with 10, 5. 2.5.
  • MCF7 transfectants were cultured in growth medium supplemented with 1.5% (v/v) FBS for 3 days in the presence or absence of tetracycline (i.e. in order to induce expression of FEZl in cells maintained in the absence of tetracycline). Thereafter, the cells were maintained in medium comprising thymidine in order to induce accumulation of cells at the Gl/S stage of the cell cycle. The thymidine-containing medium was replaced with the same growth medium, and cells were fixed at selected times thereafter. The cells were fixed in 70% ethanol and treated with propidium iodide and RNase A prior to flow-cytometry analysis.
  • Ratios were calculated as a ratio of the number of cells in the G2/M stage of the cell cycle to the number of cells in the Gl stage of the cell cycle ( Figure 8A), or as a ratio of the number of cells in the S stage of the cell cycle to the number of cells in the Gl stage of the cell cycle ( Figure 8B).
  • the results of this analysis indicate that expression of FEZl appears to inhibit MCF7 cell proliferation in vitro by causing accumulation of cells in the late S or G2/M stages of the cell cycle.
  • EEZ7 cDNA is isolated from human normal placental poly (A)+ RNA by reverse transcription polymerase-chain-reaction (RT-PCR) amplification using a pair of promoters, such as promoters having the nucleotide sequences, 5'-CAG ATG GGC AGC GTC AGT AGC CTC ATC-3' (S ⁇ Q ID NO: 58) and 5'-TCA GAT CTC AGT GGC TAT GAT GTC-3' (SEQ ID NO: 59).
  • promoters such as promoters having the nucleotide sequences, 5'-CAG ATG GGC AGC GTC AGT AGC CTC ATC-3' (S ⁇ Q ID NO: 58) and 5'-TCA GAT CTC AGT GGC TAT GAT GTC-3' (SEQ ID NO: 59).
  • any other pair of primers can be used to isolate Eez7 cDNA, or the cDNA can be made synthetically, since the sequence is now available ( Figure 5B; S ⁇ Q ID NO: 2; GenBank accession number AF 123659).
  • reverse transcription can be performed using the commercially-available SuperScript-IITM system (Gibco-BRL, catalog no. 18064-022, Rockville, MD) according to the supplier's instructions.
  • PCR can be performed, for example, using Advantage Taq (Clontech, catalog no. K1905-y) according to the supplier's instructions).
  • reverse-transcribed cDNA can be subjected to PCR amplification by maintaining a standard PCR reaction mixture at 94°C for 30 seconds, and then performing 35 cycles comprising maintaining the reaction mixture at 94°C for 10 seconds, at 58°C for 10 seconds, and at 72°C for 60 seconds, and thereafter maintaining the reaction mixture at 72°C for 60 seconds.
  • the amplified product can be separated by electrophoresis in a 1.5% (w/v) agarose gel (Gibco-BRL. catalog no. 15510-019) as described in the Cunent Protocols in Molecular Biology, ed. Frederick M Ausubel et al.. John Wiley & Sons. Inc 1987).
  • Poly (A)+ RNA can, for example, be purchased from Clontech (catalog no. 6518-1) and used to make cDNA.
  • Clontech poly (A)+ RNA material was extracted and purified from normal placenta tissue of Caucasian humans (ages 22-31) by a standard method described in Cunent Protocols in Molecular Biology (John Wiley & Sons, Inc.1987).
  • Adenoviral shuttle vector DNA can, for example, be obtained from Quantum company (Montreal, Quebec, Canada; e.g., pAdCMV-IR ⁇ S-GFP. catalog no. A ⁇ S050M).
  • Amplified EEZ7 cDNA is isolated from an agarose gel and purified using, for example, a QiagenTM PCR purification column (Stanford Valencia, CA; catalog no. 28104) according to the supplier's instructions.
  • Adenovirus shuttle vector DNA is digested using restriction endonuclease Bglll (Boehringer Mannheim-Roche; Indianapolis, IN). After the ends of the DNA are blunted using, for example. T4 DNA polymerase (Promega, Madison, WI), 10 nanograms of cDNA is ligated with 100 nanograms of vector DNA.
  • the resulting construct is used to transform an electrocompetent Escherichia coli strain, such as strain DH5a (Gibco), and the transformed cells are transfened to a culture plate containing LB agarose medium supplemented with ampicillin (e.g., as described in Cunent Protocol in Molecular Biology. John Wiley & Sons, Inc.1987).
  • an electrocompetent Escherichia coli strain such as strain DH5a (Gibco)
  • the transformed cells are transfened to a culture plate containing LB agarose medium supplemented with ampicillin (e.g., as described in Cunent Protocol in Molecular Biology. John Wiley & Sons, Inc.1987).
  • Clones which contain EEZ7 cDNA are selected, e.g. using a colony hybridization technique employing full-length EEZ7 cDNA as a DNA probe (e.g., as described in Cunent Protocol in Molecular Biology, John Wiley &. Sons. Inc.1987). These 'positive' clones are grown overnight in 5 ml of LB medium, and plasmid DNA is extracted from the positive clones, e.g. using a Qiagen miniprep column. The sequence of the extracted plasmid DNA can be analyzed at this point to confirm recovery of the anticipated construct.
  • sequencing reactions and analysis can be performed using the Applied Biosystems PrismTM BigDyeTM terminator reaction chemistry and a Perkin- ⁇ lmer Gene AmpTM PCR system 9600 and the Applied Biosystems PrismTM 377 DNA sequencing system (Norwalk CT). After confirming the orientation of the cDNA strand within the vector DNA, the plasmid can be amplified in E. coli. Confirming Transient Expression Using FEZl Adenoviral Shuttle Vectors
  • Promoter activity and adequacy of the plasmid vector can be checked by assessing transient expression of FEZl in HeLaS3 cells (ATCC) maintained in F12/MEM medium supplemented with 10 % FBS. For instance, about 5 x 10 3 cells per cubic centimeter are grown in 6- well plate overnight. Three micrograms of plasmid is used to transfect the cells in each well, for example using a lipofection method (e.g. the GenePORTERTM Reagent, Gene Therapy System Inc.). After maintaining the cells under culturing conditions (e.g. for about 48 hours), the cells are harvested and EEZ7 expression is assessed, e.g.
  • a lipofection method e.g. the GenePORTERTM Reagent, Gene Therapy System Inc.
  • Adenoviral vectors can be constructed in fetal kidney 293 cells (Microbix Biosystems Inc.. Toronto. Ontario, Canada) by transfecting the cells with the adenoviral shuttle vector described above and adenovirus DNA (e.g. obtained from Quantum), as described (Miyake et al., 1996, Proc. Natl. Acad. Sci. USA 93:1320; Kanegae et al., 1994, Jpn. J. Med. Sci. Biol. 17:157). 293 cells obtained from Microbix Biosystems Inc. are low passages and would be adequate to obtain favorable homologous recombination efficiency. Transfected 293 cells are seeded in 96-well plate, and well-isolated plaques are selected.
  • adenovirus DNA e.g. obtained from Quantum
  • 293 cells can be transfected using the shuttle plasmid by the calcium phosphate precipitation method and grown in 100 millimeter diameter dishes. Twenty- four hours following transfection, the transfectants are seeded into individual wells of a 96-well plate (containing about 200 microliters of medium per well). The cells in the well are diluted with from about 10 to 100 times the number of non-transfected 293 cells. After 2 to 3 weeks of incubation, plaque formed cells are harvested and virus particles are extracted, e.g. by multiple freeze-and-thaw cycles. The number of plaque- forming wells is estimated to about 10-50 wells per 96-well plate.
  • the virus-containing supernatant obtained from plaque-formmg wells is subjected to sequential infection of 293 cells in soft agar.
  • soft agar For example about 5 x 10 5 293 cells are infected with 100 microliters of virus-containing supernatant, and the cells are seeded in 1.25% (w/v) low-melting temperature gel (Gibco) in a 60 millimeter diameter culture dish.
  • Gibco low-melting temperature gel
  • plaques formed within the soft agar are isolated under microscopic observation.
  • GFP green fluorescent protein
  • Virus titers are propagated, for example by sequential infection of 293 cells grown in 75 to 175 milliliters of liquid culture medium in a flask.
  • FEZl in cells transfected using an adenovirus vector containing an isolated nucleic acid which encodes at least an operative portion of Fezl protein can be detected by immunoblot analysis of proteins extracted from the cell, e.g. using a rabbit anti-Fez 1 polyclonal antibody.
  • infectivity of the virus vector can be assessed by incubating HeLaS3 cells (ATCC) with an adenovirus vector- containing supernatant at a volumetric ratio of 1/40-1/10 (v/v), extracting protein from the cells, and assessing whether Fezl protein can be detected by immunoblot analysis.
  • the adenovirus vector also encodes a detectable protein such as GFP
  • infectivity of the virus vector preparation can be assessed by assessing expression of the detectable protein in the cells incubated with the virus-containing supernatant.
  • infectivity of the virus vector can be assessed by detecting fluorescence in the cells at an excitation/emission wavelength pair that is characteristic of GFP.
  • buffer A comprising 100 millimolar NaCl, 0.5% NP-40, 0.75 milligrams per milliliter bovine serum albumin (BSA), 20 millimolar Tris-HCl pH 8.0, and 1 millimolar EDTA
  • buffer B comprising 150 millimolar NaCl, 0.1% (v/v) Tween 20, 0.75 milligrams per milliliter BSA. 50 millimolar Tris-HCl pH 8.0, 5 millimolar EDTA. 10% (v/v) glycerol).
  • the beads were mixed with protein samples and washed 5 times, each wash comprising mixing the beads with 10 volumes of the binding buffer. After the beads had been washed, the bead-containing liquid was centrifuged to recover binding proteins. The samples were boiled for 3 minutes and then the proteins in the samples were separated by SDS-PAGE. The gel was dried and exposed to film for 4-24 hours at -80°C.
  • clones which encoded proteins that exhibited binding with Fezl protein were identified. When the DNA conesponding to these clones was extracted sequenced, it was found that many positive clones were redundant. Several independent clones were identified, including clones encoding peptide elongation factor 1- ⁇ (EFl- ⁇ ; cDNA sequence deposited by others as EMBL accession number X68142). EFl- ⁇ is a member of microtubule-associated protein family. To confirm the result, ⁇ -galactosidase assay was performed, and EF 1 - ⁇ exhibited strong interaction with Fezl . The reaction time was ⁇ 15 minutes, compared with a positive control reaction time of 15-20 minutes and a negative control reaction time of no reaction at >48 hours.
  • EFl- ⁇ (N) Three 35 S-metbionine-labeled deletion mutants of in vitro translated EFl- ⁇ protein were made: a mutant designated EFl- ⁇ (N) in which all but the amino- terminal 153 amino acid residues of EFl- ⁇ were deleted, a mutant designated EFl- ⁇ (C) in which all but the carboxy 1-terminal 126 amino acid residues of EFl- ⁇ were deleted, and a mutant designated EFl- ⁇ (M) in which all but 149 amino acid residues in the central portion of the EF-l ⁇ were deleted (i.e. EFl- ⁇ (M) consisted of residues 154-302, measured from the amino terminus of EFl- ⁇ ).
  • EFl- ⁇ The amino acid sequence of EFl- ⁇ can be found at GenBank accession number X68142. In vitro binding of these deletion mutants with GST-fused Fezl was analyzed. EFl- ⁇ (N) bound with Fezl, but neither EFl- ⁇ (C) nor EFl- ⁇ (M) bound with Fezl. In Vitro Binding Assav of Fezl Proteins to the Amino-Terminal Portion of EFl- ⁇ Protein.
  • the complementary binding assay was performed in buffer B using 35 S- methionine-labeled in vitro translated full-length 67 kDa Fezl (lanes 1 and 2 in Figure 12) or truncated 40 kDa Fezl protein (lanes 3 and 4 in Figure 12).
  • the assay mixtures conesponding to lanes 2 and 4 of Figure 12 contained GST fused with EFl- ⁇ (N), and the mixtures conesponding to lanes 1 and 3 of Figure 12 contained GST protein (as a negative control).
  • In vitro translated full-length 67 kDa Fezl protein (lane 5) or truncated 40 kDa Fezl protein (lane 6) were loaded alone as controls.
  • the amino acid sequence of Fezl comprises a leucine-zipper-like region. Leucine zipper regions are known to be involved in the protein-protein and/or protein-nucleotide interactions in other proteins (Proc. Natl. Acad. Sci. USA 96:3928- 3933, 1999).
  • An in vitro binding assay was performed in buffer B. wherein the assay mixtures contained either 35 S-methionine-labeled in vitro translated full-length (67 kDa) Fezl proteins (lanes 1. 2, and 5 of Figure 13) or 35 S-methionine-labeled in vitro translated truncated 40 kDa Fezl protein (lanes 3. 4, and 6).
  • the assay mixtures also contained either GST-fused full-length 67 kDa Fezl (lane 2), GST-fused truncated 40 kDa Fezl (lane 4), or GST protein (lanes 1 and 3: negative control).
  • the results of these assays indicate that the 67-kDa Fezl and truncated 40-kDa Fezl proteins can dimerize.
  • Interaction of Fezl with EFl- ⁇ in Transfected Cells Full-length FEZl cDNA was ligated with pcDNAV5 vector (Invitrogen,
  • Lanes 1 and 7 in Figure 14 represent vector control transfectant lysates, in which neither tag could be detected.
  • IP immunoprecipitation experiments
  • NRS normal serum
  • a rabbit polyclonal antibody which binds specifically with human Fezl has been developed. Specificity of binding of the polyclonal antibody for Fezl protein was demonstrated as follows. FEZl cDNA was ligated with a GST-fusion expression vector (pGEX, Pharmacia), and the protein was expressed in E. coli cells and purified. The Fezl -GST fusion protein was inoculated into rabbits to raise the anti-Fez 1 antibody, which was harvested according to standard methods.
  • Figure 13A shows the results of an immunoblot analysis performed using the polyclonal anti-Fezl antibody.
  • Lanes 1-3 or 50(lanes 4-6) micrograms of protein obtained from human brain (lanes 1 and 4), testis (lanes 2 and 5), and spleen (lanes 3 and 6) were blotted onto a surface. Longer exposure of the film showed faint expression of Fezl in testis and spleen. Lane 7 contained in vitro translated full-length Fezl protein, and lane 8 contained in vitro translated truncated Fezl protein (i.e. lacking the C-terminal portion).
  • Figure 13B shows the results of an immunoprecipitation assay performed using the polyclonal anti-Fezl antibody.
  • HeLaS3 cells which do not express EEZ7, were transfected with EEZ7 cDNA ligated into expression vector pcDNA (Invitrogen) in frame with a V5 tag sequence. The cells were lysed, and the lysate was immunoprecipitated with polyclonal anti-Fezl antibody (lane 1) or with the pre-immune normal rabbit serum (lane2). The precipitates were blotted and probed using the anti-V5 tag antibody. Standard methods can be used to construct one or more monoclonal antibodies which bind specifically with Fezl protein.
  • Example 6 Post-Translational Modification of Fezl Protein
  • Cells of MCF7 clone 54 were cultured in tetracycline-free medium containing aphidicolin and either 10%> (v/v) FBS or no FBS in order to synchronize cell cycles.
  • the medium was replaced with aphidicolin-free medium with 10% serum, and the cells were incubated for the periods indicated in Figures 16A and 16B.
  • cell lysates were obtained, and the lysates were subjected to immunoblot analysis using the rabbit anti-Fezl polyclonal antibody or with an anti-actin monoclonal antibody.
  • the results of this experiment demonstrated that cellular Fezl protein is post-translationally modified in a cell cycle progression- dependent manner.
  • Fetal kidney 293 cells (which express FEZl) were maintained in serum- free medium containing aphidicolin in order to synchronize cell cycles. At a selected time, the medium was replaced with aphidicolin-free medium containing 10% (v/v) FBS, and the cells were incubated for the times indicated in Figure 17, after which incubation cellular proteins were extracted. The extracted proteins were subjected to immunoblot analysis using rabbit anti-Fezl polyclonal antibody or with an anti-actin monoclonal antibody. The results of this experiment are depicted in Figure 17. The MCF7/Fezl transfectant lysate which were used in the experiments for which results are depicted in Figure 16A were separated by SDS-PAGE in the presence of 6 molar urea.
  • Cytoplasmic and nuclear protein samples were prepared as the followings. Cytoplasmic and nuclear protein were isolated as described (DNA 7:47-55. 1998) with minor modifications. Briefly, about 10 7 293 cells were harvested and washed with PBS (10 millimolar NaPO 4 pH 7.4, 150 millimolar NaCl). After sedimenting the cells, the packed cell volume (PCV) was measured and the cells were re-suspended in 3 PCVs of freshly prepared hypotonic buffer (10 millimolar HEPES pH 7.9, 0.75 millimolar spermidine, 0.15 millimolar spermine, 0.1 millimolar EDTA, 0.1 millimolar EGTA, 1 millimolar DTT, 10 millimolar KC1). The cells were allowed to swell for 10 minutes at about 0°C. and were centrifuged at 300 x g for 10 min at 4°C. The supernatant was collected as cytoplasmic extract I (Cl).
  • PBS millimolar NaPO 4 pH 7.4, 150 millimolar NaCl
  • the pellet was re-suspended with 2.9 PCVs of hypotonic buffer.
  • the cells were broken by ten strokes using a Dounce homogenizer (Kontes Glass Co.).
  • One volume of Sucrose restore buffer (prepared by adding 9 volumes of 75% sucrose to 1 volume of lOx salts) was added and was homogenized with 10 additional strokes of the homogenizer.
  • the composition of lOx salts was as follows: 500 millimolar HEPES pH 7.9, 7.5 millimolar spermidine, 1.5 millimolar spermine. 100 millimolar KC1, 2 millimolar EDTA, 10 millimolar DTT.
  • the homogenate was centrifuged for 30 seconds at 10,000 rotations per minute in a Sorvall HB-4 rotor (16,000 x g) at 4°C. The supernatant was collected as cytoplasmic extract II (C2).
  • the pellet was re-suspended in nuclear re-suspension buffer, using about 3 milliliters per 10 9 cells.
  • Nuclear re-suspension buffer comprises 9 volumes of 20 millimolar HEPES pH 7.9, 0.75 millimolar spermidine, 0.15 millimolar spermine, 0.2 millimolar EDTA, 2 millimolar EGTA, 2 millimolar DTT, 25% (v/v) glycerol and 1 volume of a (4°C) saturated solution of ammonium sulfate.
  • the re-suspended pellet was incubated for about 30 minutes at 4°C with occasional rocking.
  • the extract was sedimented by centrifugation at 4°C for 120 minutes at 150.000 x g.
  • Nuclear dialysis buffer comprises 20 millimolar HEPES pH 7.9, 20% (v/v) glycerol, 100 millimolar KC1. 0.2 millimolar EDTA, 0.2 millimolar EGTA, 2 millimolar DTT).
  • the nuclear extract (N) was stored at -80°C.
  • Example 8 Interaction of Fezl with Microtubules Cytoplasmic protein fractions were obtained from Fezl -expressing 293 cells ("Tax” in Figure 22) which had been incubated with paclitaxel in order to polymerize tubulin and from Fezl -expressing 293 cells ("Col” in Figure 22) which had been incubated with colchicine for non-polymerization (i.e. as a control).
  • the 293 cells were selected from three groups: non-synchronized cells ("non-treatment” in Figure 22), Gl/S-synchronized cells ("0 h” in Figure 22), and S-to-G2/M-synchronized cells ("8 h” in Figure 22).
  • Example 9 A Proposed Biological Function for Fezl Protein It is recognized that the characteristics described herein for Fezl proteins and nucleic acids which encode them do not depend on the accuracy or reliability of any theories presented in this Example with regard to the physiological function of Fezl protein. Thus, without being bound by any particular theory of operation, the inventors propose the following biological functions for Fezl protein. Immunoblot analysis of extracts obtained from cells which express
  • EEZ7 demonstrates that Fezl protein is predominantly localized in the cytoplasm, but is also found in the nucleus.
  • Yeast two-hybrid screening demonstrates that at least one peptide elongation factor ( ⁇ Fl- ⁇ ) is a likely binding partner of Fezl protein.
  • ⁇ Fl- ⁇ peptide elongation factor
  • Others have discovered that the ⁇ F family of proteins not only function as a peptide chain elongation factors, but are involved in interactions between microtubules and in the process of tubulin polymerization (see. e.g., ⁇ ur. J. Biochem. 171 :119. 1988; Proc. Natl. Acad. Sci. USA 90:3028, 1993; Plant Cell 6:893, 1994; Cell Motil. Cytoskel. 41 : 168.
  • Fezl was determined not to be associated with depolymerized microtubule precipitates in the presence of the tubulin polymerization inhibitor colchicine.
  • the data presented in this application indicate that Fezl protein serves to modulate polymerization and stability of microtubules, and possibly other cytoskeletal features, in vivo.
  • Fezl protein can be expected to be involved in cellular processes which are modulated by cytoskeletal stability and changes. Examples of such cellular processes include initiation of mitosis, modulation of the rate and stage of mitosis, modulation of the initiation and rate of cell proliferation and growth, modulation of cell shape and rigidity, modulation of cell motility.
  • modulation of the rate and stage of cellular DNA replication modulation of the intracellular distribution of organelles (e.g. mitochondria, endoplasmic reticulum, Golgi apparatus, chloroplasts, and the like), modulating the metastatic potential of a cell, and modulation of cellular transformation from a non-cancerous to a cancerous phenotype.
  • organelles e.g. mitochondria, endoplasmic reticulum, Golgi apparatus, chloroplasts, and the like
  • modulating the metastatic potential of a cell e.g. mitochondria, endoplasmic reticulum, Golgi apparatus, chloroplasts, and the like
  • modulating the metastatic potential of a cell e.g. a cell division from a non-cancerous to a cancerous phenotype.
  • cell division of higher eukaryotes is known to be initiated and be regulated according to a dynamic process, which involves the so-called mitotic apparatus (an organized complex of proteins) that distribute the duplicated
  • the extended microtubular cytoskeleton of an inte ⁇ hase cell is disassembled into tubulin subunits, and. when an appropriate point in cell cycle occurs, the tubulin subunits are re-assembled into two sets of polarized spindle tubes, that function as a central part of the mitotic apparatus.
  • the growing tubules attached at an end of a condensed chromosome.
  • the tubules meet or attach at a collection of proteins designated the centrosome or microtubule organellar center.
  • the centrosome complex has been isolated by others (see. e.g., Telzer, 1979, J. Cell Biol.
  • the centrosome comprises ⁇ -, ⁇ - and ⁇ -tubulin. heat shock protein 70, and an elongation factor protein (Eur. J.
  • tubulin is a target for known anti-cancer drugs, such as paclitaxel (which can induce tubulin polymerization) and vinca alkaloids (which can inhibit polymerization process; Med. Res. Rev. 18:259-296, 1998).
  • paclitaxel which can induce tubulin polymerization
  • vinca alkaloids which can inhibit polymerization process
  • Other known tumor suppressor genes have been shown to be involved in the dynamics of microtubule assembly and disassembly.
  • APC can promote microtubules assembly (Eur. J. Biochem.
  • Fezl can inhibit tubulin polymerization. Because, as demonstrated herein, Fezl binds with at least one EF protein, and because these proteins have been identified as a soluble protein component from the centrosome, it can be expected that Fezl has an role in the late events of the cell division process or centrosomal dynamics. This is in keeping with the finding herein that Fezl protein induces accumulation of cells in the late S to G2/M stage(s) of the cell cycle. In these stages the centrosome is undergoing assembly in daughter cells.
  • the experiments described herein demonstrate at least two ways in which the activity of Fezl can be affected, namely by phosphorylation of Fezl protein and by binding a polypeptide or polypeptide-like molecule with Fezl protein.
  • the results presented herein demonstrate that phosphorylation of Fezl by PKA can diminish the ability of Fezl to inhibit tubulin polymerization.
  • Agents which directly phosphorylate Fezl or which induce its phosphorylation or inhibit its dephosphorylation by other proteins are useful for diminishing the ability of Fezl to inhibit tubulin polymerization and conesponding growth/shrinkage and maintenance of cytoskeletal features (e.g. microtubules) which contain tubulin or tubulin-like proteins.
  • Agents which directly dephosphorylate Fezl or which induce its dephosphorylation or inhibit its phosphorylation by other proteins are useful for enhancing the ability of Fezl to inhibit polymerization and conesponding growth/shrinkage and maintenance of cytoskeletal features which contain tubulin or tubulin-like proteins.
  • Agents which are able to bind specifically with Fezl protein can also modulate its physiological activity. Examples of such agents are antibodies which are raised against Fezl protein, tubulin, and EFl- ⁇ . Fragments of such proteins (e.g. Fc portions of antibodies or the EFl- ⁇ (N) fragment described herein) can exhibit effects on Fezl protein that are similar to the effects of the whole protein on Fezl protein.
  • peptide or peptidomimetic compounds which mimic the structure of the portion of a protein that binds specifically with Fezl protein can exhibit effects on Fezl protein that are similar to the effects of the conesponding whole protein on Fezl protein.
  • numerous methods known in the art can be used to construct and screen libraries of compounds which are structurally similar to proteins that bind specifically with Fezl protein (e.g. peptide or peptidomimetic compounds which are structurally similar to one or more portions of tubulin, EF 1 - ⁇ , or an antibody that binds specifically with Fezl).
  • Fezl protein appears to form dimers or multimers indicates that compounds which are identical to or which mimic the structure of a portion of Fezl involved in dimerization or multimerization can also be used to modulate the physiological activity of Fezl.
  • methods of constructing and screening libraries of compounds which are identical to or structurally similar to a dimerization/multimerization domain e.g. a library including random fragments of Fezl protein

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Abstract

L'invention concerne des homologues de polynucléotides isolés avec une partie d'un brin du gène suppresseur de tumeur de l'homme, le gène FEZ1. L'invention traite également de la protéine suppresseur de tumeur ainsi codée, Fezl. Ces polynucléotides présentent un grand intérêt, par exemple, comme sondes, comme amorces, comme parties de vecteurs d'expression et similaires. L'invention traite également de procédés de diagnostique, de thérapie, de prolifération cellulaire et de dosage utilisant ces polynucléotides et ces protéines. L'invention a également pour objet des kits permettant de mettre en oeuvre les procédés selon l'invention.
EP00912007A 1999-02-25 2000-02-25 Compositions, kits et procedes concernant le gene fez1 de l'homme, nouveau gene suppresseur des tumeurs Withdrawn EP1163252A4 (fr)

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US7141417B1 (en) 1999-02-25 2006-11-28 Thomas Jefferson University Compositions, kits, and methods relating to the human FEZ1 gene, a novel tumor suppressor gene
EP2167521A4 (fr) 2007-06-15 2011-11-23 Univ Ohio State Res Found Protéines de fusion all-1 oncogènes pour cibler le traitement de micro-arn régulé par drosha
EP2657353B1 (fr) 2007-08-03 2017-04-12 The Ohio State University Research Foundation Régions ultraconservées codant de l'ARNnm
JP2011504093A (ja) 2007-10-26 2011-02-03 ジ・オハイオ・ステイト・ユニバーシティ・リサーチ・ファウンデイション 脆弱性ヒスチジン三連構造（ｆｈｉｔ）相互作用を同定するための方法およびその使用
ES2433940T3 (es) 2008-06-11 2013-12-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Uso de la familia miR-26 como marcador predictivo del carcinoma hepatocelular y sensibilidad a la terapia
EP2504452A4 (fr) 2009-11-23 2014-06-11 Univ Ohio State Res Found Substances et procédés pouvant s'utiliser pour agir sur la croissance, la migration, et l'invasion de cellules tumorales
CA2816603A1 (fr) 2010-11-12 2012-05-18 The Ohio State University Research Foundation Materiaux et procedes relatifs aux microarn-21, reparation de desappariement et cancer colorectal
CN103313706A (zh) 2010-11-15 2013-09-18 俄亥俄州立大学研究基金会 控制释放粘膜粘合系统
CA2828772A1 (fr) 2011-03-07 2012-09-13 The Ohio State University Activite mutatrice induite par l'inflammation des liaisons au microarn-155 (mir-155) et le cancer
EP2766500A4 (fr) 2011-10-14 2015-10-14 Univ Ohio State Méthodes et matériaux relatifs au cancer des ovaires
AU2012352265B2 (en) 2011-12-13 2017-02-16 Ohio State Innovation Foundation Methods and compositions related to miR-21 and miR-29a, exosome inhibition, and cancer metastasis
CA2866052A1 (fr) 2012-01-20 2013-07-25 The Ohio State University Signatures de marqueurs biologiques du cancer du sein concernant le pouvoir envahissant et le pronostic

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WO2001055448A1 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps

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WO2001055300A2 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2001055448A1 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps

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ISHII H ET AL: "The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. UNITED STATES 30 MAR 1999, vol. 96, no. 7, 30 March 1999 (1999-03-30), pages 3928-3933, XP002266793 ISSN: 0027-8424 *
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